Synthetic lethality and DNA damage response targeting in cancer stem cells: a comprehensive review.

Introduction

Cancer remains a formidable global health challenge, characterized by uncontrolled cell proliferation, evasion of apoptosis, and metastatic dissemination [ 1 ]. Despite significant advancements in conventional treatments, including surgery, chemotherapy, and radiation therapy, tumor recurrence, metastasis, and acquired drug resistance continue to impede long-term patient survival [ 1 ]. A pivotal factor contributing to these therapeutic failures is the presence of a distinct subpopulation of cells within tumors known as Cancer Stem Cells (CSCs), also referred to as tumor-initiating cells (TICs) [ 1 ]. CSCs possess hallmark stem cell properties, including indefinite self-renewal, multilineage differentiation potential, and the capacity to regenerate heterogeneous tumors [ 1 ]. While their frequency can vary, CSCs often represent a minor subset of tumor cells [ 2 ]. The origin of CSCs is not fully understood, but they may arise from normal stem cells, committed progenitors, or even differentiated cells that undergo dedifferentiation or trans-differentiation processes [ 2 , 3 ]. Their unique biological characteristics, such as quiescence, enhanced DNA repair mechanisms, and active drug efflux pumps, confer intrinsic resistance to conventional therapies, leading to their enrichment post-treatment and subsequent tumor relapse [ 2 , 4 ]. Therefore, selective targeting and eradication of CSCs are considered paramount for achieving durable therapeutic responses and preventing disease progression [ 1 , 5 ].

The maintenance of genomic integrity is fundamental for cellular viability and preventing malignant transformation, a process meticulously overseen by the DNA Damage Response (DDR) pathways [ 6 ]. These intricate networks detect DNA lesions arising from endogenous processes or exogenous genotoxic insults, orchestrating their repair, regulating cell cycle progression, and dictating cell fate decisions [ 6 , 7 ]. However, dysregulation and hyperactivity of DDR pathways are characteristic hallmarks of most cancer cells, contributing to genomic instability while simultaneously conferring survival advantages and therapeutic resistance [ 8 ]. Notably, CSCs often exhibit an enhanced DDR capacity, making them particularly adept at repairing chemotherapy- and radiotherapy-induced DNA damage, thereby promoting their survival [ 9 ].

In this context, the genetic concept of Synthetic Lethality (SL) offers a powerful and selective therapeutic paradigm [ 10 ]. SL occurs when the combined inactivation of two genes or molecular processes leads to cell death, while the individual inactivation of either is compatible with cell viability [ 11 ]. This principle can be exploited to selectively target cancer cells that harbor specific genetic or functional defects, such as those in DDR pathways, by inhibiting a compensatory or parallel pathway [ 12 , 13 ]. The clinical success of Poly (ADP-ribose) polymerase (PARP) inhibitors in BRCA -deficient cancers exemplify the therapeutic potential of SL [ 14 ].

This review aims to provide a comprehensive overview of the current understanding and advancements in targeting CSCs through the lens of synthetic lethality and DDR. The discussion will delve into the unique vulnerabilities of CSCs, the mechanisms by which DDR pathways contribute to their survival and resistance, and the development of novel therapeutic strategies, including small molecule inhibitors, antibody-drug conjugates, and advanced cell therapies. Furthermore, a critical examination of the challenges in translating these promising approaches to clinical practice will be presented, highlighting the emerging role of integrated diagnostic platforms in guiding personalized cancer treatment.

Cancer stem cell biology and its role in therapeutic resistance

Defining and identifying cancer stem cells (CSCs)

Cancer stem cells (CSCs), also known as tumor-initiating cells (TICs), represent a distinct subpopulation within heterogeneous tumors that possess unique stem-like properties crucial for tumor initiation, growth, metastasis, and therapeutic resistance [ 1 , 15 ]. These properties include self-renewal, the capacity to generate differentiated progeny that constitute the bulk of the tumor, and the ability to initiate tumor formation upon serial transplantation into immunocompromised mice [ 1 ]. While their frequency can vary, CSCs often represent a minor subset of tumor cells [ 2 , 16 ]. The origin of CSCs is not fully understood, but they may arise from normal stem cells, committed progenitors, or even differentiated cells that undergo dedifferentiation or trans-differentiation processes [ 2 ].

Identification and isolation of CSCs primarily rely on specific cell-surface markers, often shared with corresponding normal stem cells, or functional assays [ 17 ] (Table 1 ).

The identification of CSCs, however, presents a significant challenge due to the dynamic nature of their marker expression and the concept of phenotypic plasticity. While a wide array of CSC markers has been identified (Table 1 ), the field is moving away from a rigid, hierarchical model defined by static markers. Recent studies (2020–2024) emphasize that “stemness” is often a transient, dynamic state that non-CSCs can acquire in response to TME cues or therapeutic pressure [ 18 ]. This means that many “markers” are not simply identifiers but are functional drivers of the stem state; for example, LGR5 is a critical Wnt-pathway component, and high ALDH activity reflects a specific metabolic state. This plasticity implies that diagnostic and therapeutic strategies must be adaptive, perhaps targeting the drivers of the stem state rather than just the surface markers.

Commonly used markers include CD44 (CD44+/CD24- in breast cancer [ 19 ], CD44v6 in colorectal cancer), CD133 (in brain tumors, colorectal, pancreatic, liver, skin, prostate, ovarian cancers) [ 20 , 21 ], ALDH (aldehyde dehydrogenase activity, particularly ALDH1A1), and the side population (SP) phenotype based on Hoechst dye exclusion [ 1 , 22 ]. Other markers like EpCAM, CD166, LGR5, GPC3, B7-H3, MUC1, Lewis Y, Globo H, GD2, and gangliosides (e.g., GD3) are also being investigated [ 23 ].

The identification of CSCs presents a challenge due to the dynamic nature of their marker expression. While a wide array of CSC markers has been identified across various cancer types, inconsistencies in their expression and the ability of CSCs to undergo phenotypic changes mean that they are not a static population defined by a universal set of markers [ 1 ]. This necessitates a shift towards identifying functional characteristics or context-dependent marker combinations rather than relying on single, fixed biomarkers. This also implies that diagnostic and therapeutic strategies must be adaptive and potentially personalized, moving beyond traditional “one-size-fits-all” approaches.

The integrated CSC resistance network

CSCs are inherently resistant to conventional anti-cancer therapies, which largely target rapidly proliferating cells, while CSCs often reside in a quiescent (G0) state, making them less susceptible to cytotoxic agents [ 24 ] This quiescence allows them to survive treatment and re-initiate tumor growth post-therapy [ 25 ]. This characteristic of quiescence, while a defense mechanism, also presents a potential therapeutic vulnerability. Strategies that can either directly target quiescent cells or induce them into a proliferative state before administering conventional cytotoxic agents could be crucial for achieving complete eradication of the CSC reservoir.

Key mechanisms contributing to CSC resistance include:

Enhanced DNA Damage Response (DDR) and Repair: CSCs possess a highly efficient DDR system and enhanced DNA repair capacity, allowing them to rapidly repair chemotherapy- and radiotherapy-induced DNA lesions [ 24 , 26 ]. This includes robust activation of cell cycle checkpoints and elevated homologous recombination (HR)-mediated DNA repair [ 27 ]. Increased Drug Efflux: CSCs often overexpress ATP-binding cassette (ABC) transporters (e.g., MDR1, ABCG2), which actively pump out chemotherapeutic drugs, reducing intracellular drug concentrations and promoting multi-drug resistance [ 2 , 28 ]. Altered Metabolism (Metabostemness): CSCs exhibit metabolic plasticity, adapting their energy production to survive harsh microenvironmental conditions and resist therapies. This includes reliance on glycolysis, oxidative phosphorylation (OXPHOS), and altered amino acid and lipid metabolism [ 29 , 30 ]. Epithelial-Mesenchymal Transition (EMT): EMT confers stem-like features, increased invasiveness, and drug resistance to cancer cells. CSCs often exhibit a mesenchymal phenotype or can undergo EMT, contributing to their resistance and metastatic potential [ 24 , 31 ]. Immunosuppressive Tumor Microenvironment (TME): CSCs actively interact with and remodel their niche, creating an immunosuppressive environment that shields them from immune surveillance and therapies. This involves secreting immunosuppressive cytokines (e.g., TGF-β, IL-10) [ 32 ], recruiting immune cells like M2 macrophages and myeloid-derived suppressor cells (MDSCs), and upregulating immune checkpoint molecules (e.g., PD-L1, B7-H3) [ 24 ].

The multifaceted nature of CSC resistance, encompassing DNA repair, drug efflux, metabolic adaptations, EMT, and TME modulation, suggests that these mechanisms are not isolated but rather form a complex, interconnected network. For instance, EMT can induce stem-like features and drug resistance [ 33 ], and TME factors can directly influence CSC properties and resistance [ 24 ]. This implies that monotherapies targeting a single resistance pathway are likely to be insufficient due to compensatory mechanisms or the activation of alternative resistance routes [ 34 ]. Effective CSC eradication will therefore require multi-modal or combination therapies that simultaneously target multiple, interconnected resistance pathways, or strategies that disrupt the “hub” mechanisms underlying these interconnections (Fig. 1 ).

Fig. 1 Mechanisms of cancer stem cell (CSC) resistance. CSCs often remain in a quiescent (G0) state, enabling survival after therapy and tumor relapse. Major resistance mechanisms include: (i) enhanced DNA damage response and repair, (ii) increased drug efflux via ABC transporters, (iii) altered metabolism (glycolysis, OXPHOS, amino acid and lipid pathways), (iv) epithelial-mesenchymal transition (EMT), and (v) remodeling of an immunosuppressive tumor microenvironment. These interconnected mechanisms collectively contribute to CSC survival, therapy resistance, and disease progression (Images were drawn by AI and further edited with adobe illustrator)

Table 1 Key cancer stem cell markers and their clinical relevance Marker Associated cancer types Functional roles in CSCs Resistance mechanisms Therapeutic relevance/status References CD44 Breast (CD44+/CD24-), Colorectal (CD44v6), Prostate, Ovarian, Head & Neck, Pancreatic, Lung, Myeloid Leukemia, Multiple Myeloma Self-renewal, tumor initiation, migration, invasion, metastasis, EMT, adhesion, signaling platform (integrates microenvironmental cues, growth factors, cytokines), regulates PI3K/AKT, STAT3, Ras-MAPK pathways Chemo/radioresistance, drug efflux, EMT, oxidative stress resistance, epigenetic control Biomarker for isolation/enrichment, prognostic marker. CD44v6 CAR-T in Phase 2 trials [ 2 ] CD133 Brain (Glioblastoma), Colorectal, Pancreatic, Liver, Skin, Prostate, Ovarian, Lung, Head & Neck Self-renewal, clonogenic capacity, tumor initiation, differentiation, migration, invasion, survival under stress, associated with pluripotency genes (Oct4, Sox2, Nanog, c-Myc), forms signaling platforms (PI3K/Akt, Wnt/β-catenin, Src) Chemoresistance, radioresistance, drug efflux, immune evasion, promotes EMT Widely used CSC marker. CAR-T/NK cell therapies targeting CD133 in preclinical/clinical trials [ 1 ] ALDH1A1 Breast, Head & Neck, Lung, Pancreatic Self-renewal, tumorigenicity, metabolizes genotoxic aldehydes, modulates energy metabolism Chemo/radioresistance Potential target for immunotherapy 26 EpCAM Breast, Prostate, Ovarian, Colorectal, Pancreatic, Head & Neck, Oral, Lung Cell proliferation, migration, differentiation, cell adhesion, CSC self-renewal, influences Wnt/β-catenin, EGFR pathways, metabolic reprogramming, epigenetic regulation Tumorigenicity, therapeutic resistance, EMT, immune evasion Target for monoclonal antibodies, ADCs, CAR T/NK cell therapies. Clinical trials ongoing [ 35 ] MUC1 Breast, Prostate, Pancreatic, Lung, Ovarian, Gastric, Colorectal, Oral, Leukemia Self-renewal, proliferation, metastasis, angiogenesis, invasion, regulates Wnt/β-catenin, NF-κB, PI3K-AKT, MAPK pathways Drug resistance, immune evasion (aberrant glycosylation) Target for ADCs, CAR-T cells, radiopharmaceuticals, vaccines. Clinical trials ongoing [ 36 ] GD2 Neuroblastoma, Melanoma, Glioma, Retinoblastoma, Osteosarcoma, Ewing’s Sarcoma, Rhabdomyosarcoma, Small Cell Lung Cancer, Breast Cancer Tumor-associated surface antigen, promotes cell proliferation, motility, migration, adhesion, invasion Associated with stem cells (neural, mesenchymal, breast CSCs) Standard of care for high-risk neuroblastoma (anti-GD2 antibodies), target for ADCs, CAR-T/NKT cells. Clinical trials ongoing [ 37 ] GD3 Glioblastoma, Breast Cancer Promotes cell growth, plasticity, clonogenicity, tumorigenicity, migration Chemotherapy resistance (Temozolomide) Potential therapeutic target, especially via ST8SIA1 inhibition [ 38 ] Lewis Y Small Cell Lung Cancer Blood group antigen, potential target for antibody-based immunotherapy – Hu3S193 monoclonal antibody in pilot trials [ 37 ] Globo H Breast, Gastric, Pancreatic, Lung, Colorectal, Ovarian, Prostate Tumor-associated glycan – Target for ADCs (OBI-999) and vaccines. Phase 1/2 trials ongoing [ 39 ] GPNMB Breast, Glioblastoma, Lung, Laryngeal Tumor initiation, malignant progression, EMT induction, acquisition of stem-like properties, promotes migration, invasion, sphere/tumor formation, interacts with EGFR, activates STAT3 signaling Associated with dormant cancer cells, chemoresistance Potential diagnostic marker and therapeutic target LGR5 Colorectal, Gastrointestinal Marks putative CSCs, overexpressed in various GI cancers, Wnt agonist receptor – Target for ADCs (anti-LGR5-vc-MMAE, anti-LGR5-NMS818), BiTEs, CARs in preclinical models [ 40 ] GPC3 Hepatocellular Carcinoma (HCC), Hepatoblastoma, Embryonal Sarcoma, Malignant Rhabdoid Tumor, Yolk Sac Tumor, Wilm’s Tumor, Squamous Cell Carcinoma of Lung, Liposarcoma Enhances Wnt signaling, stimulates tumor growth, increases cell proliferation – Attractive target for immunotherapy (GC33 mAb, CAR-T cells). Clinical trials ongoing [ 41 ] B7-H3 (CD276) Glioblastoma, Ovarian, Liver, Prostate, Chordoma, various solid tumors Immunoregulatory ligand, correlates with tumor progression/metastasis, decreases immune cell infiltration, downregulates lymphocyte responses Promotes tumorigenicity, associated with radiation resistance Potential target for CAR-T cell therapy. Clinical trials ongoing. Small molecule FUT8 inhibitor (FDW028) targets B7-H3 [ 42 ] CD166 (ALCAM) Breast, Colorectal, Prostate, Melanoma, Pancreatic Transmembrane glycoprotein, involved in hematopoiesis, neurogenesis, inflammatory responses, associated with tumorigenesis – Target for CAR-T cells in preclinical studies [ 43 ] PSCA Prostate, Pancreatic Prostate stem cell antigen – Target for CAR-T cells. Clinical trials ongoing [ 44 ] CD117 Prostate Associated with heightened PCa aggressiveness Fosters CSC traits, resistance to tyrosine kinase inhibitors Potential target for CAR-engineered cells [ 45 ] Tn Antigen Majority of human carcinomas (e.g., breast, cervical, lung, colorectal, gastric) Tumor-associated carbohydrate antigen, arises from blocked O-glycosylation, correlates with metastatic potential and poor prognosis May influence cell adhesion, migration, metastasis, immune evasion Target for antibodies, vaccines, CAR-T cells. Clinical trials ongoing [ 46 ] Sialyl Lewis X (sLeX) Head & Neck, GI carcinomas, Breast, Colorectal, Prostate, Lung Tetrasaccharide carbohydrate, promotes metastatic dissemination (binding to E-selectin), associated with tumor invasion, influences angiogenesis, immune recognition – Prognostic marker, target for monoclonal antibodies. Clinical trials ongoing [ 47 ]

DNA damage response (DDR) pathways in cancer

Fundamental mechanisms of DNA repair and checkpoints

The maintenance of genomic integrity is paramount for cellular function and survival, a process meticulously orchestrated by the DNA Damage Response (DDR) pathways [ 48 ]. These complex networks detect various forms of DNA lesions, activate cell cycle checkpoints to halt progression, and initiate DNA repair mechanisms to restore genomic fidelity [ 6 , 49 ]. Key DNA repair pathways include:

Nucleotide Excision Repair (NER): This pathway repairs bulky, helix-distorting lesions caused by UV light, exogenous chemicals, and certain chemotherapeutic agents like cisplatin [ 6 ]. Base Excision Repair (BER): This mechanism addresses single-base damage (e.g., oxidation, alkylation, deamination) and single-strand breaks (SSBs) [ 6 ] Poly(ADP-ribose) polymerase (PARP) is a crucial enzyme in BER, binding to SSBs and recruiting repair factors [ 50 ]. Mismatch Repair (MMR): The MMR system corrects base mismatches and small insertion/deletion loops that arise during DNA replication [ 6 , 51 ]. Homologous Recombination (HR): This is an error-free pathway that repairs DNA double-strand breaks (DSBs) by using a homologous DNA template, typically a sister chromatid [ 6 ] BRCA1 and BRCA2 are central to HR [ 14 ]. Non-Homologous End-Joining (NHEJ): This is a more error-prone pathway for DSB repair that directly ligates broken DNA ends without a homologous template [ 6 , 52 ] DNA-dependent protein kinase catalytic subunit (DNA-PKcs) is a key component of NHEJ [ 53 ].

Cell cycle checkpoints (e.g., G1, S, G2/M) are surveillance mechanisms that monitor DNA integrity and replication completion, pausing the cell cycle to allow time for repair or inducing apoptosis/senescence if damage is irreparable [ 6 , 54 ]. Key kinases involved include ATM (Ataxia Telangiectasia Mutated), ATR (ATM and Rad3-related), Chk1, and Chk2 [ 6 ] (Fig. 2 ).

Fig. 2 Fundamental mechanisms of DNA repair and checkpoints. The DNA damage response (DDR) engages multiple repair pathways, including nucleotide excision repair (NER), base excision repair (BER), mismatch repair (MMR), homologous recombination (HR), and non-homologous end-joining (NHEJ). Cell cycle checkpoints (G1, S, G2/M) are coordinated by key kinases such as ATM, ATR, CHK1, and CHK2, which pause the cycle to allow repair or trigger apoptosis if damage is irreparable. Together, these pathways safeguard genomic integrity and maintain cellular homeostasis (Images were drawn by AI and further edited with adobe illustrator)

Dysregulation and hyperactivity of DDR in cancer cells and CSCs

Genomic instability is a hallmark of cancer, often stemming from dysregulated DDR pathways [ 6 ] While defects in DDR can predispose to cancer, cancer cells frequently exhibit hyperactive DDR mechanisms to cope with increased endogenous replication stress (due to oncogene activation) and to survive genotoxic therapies [ 8 , 55 ].

Oncogene-induced replication stress: Oncogenes (e.g., Ras, Myc, Cyclin E) disrupt normal cell cycle regulation, causing replication stress and increasing reliance on ATR-Chk1 pathway for survival [ 56 ]. Constitutive ATM activation: Self-inflicted DNA DSBs in cancer cells can lead to persistent ATM activation, which, unexpectedly, sustains tumorigenicity and stemness rather than suppressing them [ 57 ]. This process involves a cascade initiated by low-level cytochrome c leakage, sublethal caspase/nuclease activation, and subsequent activation of NF-κB and STAT3 [ 57 ]. This represents a paradoxical shift in function for ATM, from a tumor suppressor to a mechanism that promotes cancer cell survival and stemness [ 58 ]. This duality implies that simply enhancing DDR might not always be beneficial in cancer treatment [ 58 ]. Instead, precision strategies must aim to disrupt the aberrant DDR mechanisms that cancer cells, especially CSCs, exploit for survival, or to re-sensitize them to DNA damage by inhibiting these hyperactive pathways. This understanding is crucial for designing therapies that selectively target cancer vulnerabilities without harming normal cells [ 59 ]. Elevated DNA repair capacity in CSCs: CSCs often display a more robust DDR compared to bulk tumor cells, leading to improved repair of cytotoxic DNA lesions and increased survival [ 50 ] This enhanced repair contributes significantly to their resistance to chemo- and radiotherapy [ 50 , 60 ].

Reliance of CSCs on specific DDR pathways

CSCs exhibit a heightened reliance on specific DDR pathways for their survival and resistance, making these pathways attractive therapeutic targets.

Homologous Recombination (HR): Breast cancer stem cells (BCSCs) and glioblastoma stem cells (GSCs) show elevated HR activity, contributing to their radioresistance [ 27 ] RAD51, a key HR protein, is often overexpressed in CSCs [ 27 ]. Replication Stress (RS) Response (ATR-CHK1): CSCs display an effective RS response, with the ATR-CHK1 signaling pathway being crucial for genome stability in BCSCs [ 27 ]. GSCs also show elevated expression of CHK1 and ATR, leading to robust activation of the G2/M checkpoint after irradiation [ 27 , 61 ]. Non-Homologous End-Joining (NHEJ): The error-prone NHEJ pathway, particularly DNA-PK activity, can act as a therapeutic barrier in CSCs. DNA-PKcs is highly expressed in GSCs, and its inhibition reduces tumor stemness and sensitizes xenografts to radiotherapyb [ 27 ]. PARP1: Overexpression of PARP1 has been observed in colorectal CSCs, influencing their phenotype depending on p53 status [ 62 ]. While its role in stemness maintenance is complex, PARP1 is a key player in DNA repair and can be hyperactivated in metabolically active cancer cells [ 63 ].

The robust DDR in CSCs, while a source of resistance, also creates a dependency on these specific pathways (e.g., HR, ATR-CHK1, NHEJ) for survival [ 27 ]. If these pathways are inhibited, CSCs become vulnerable. This dependency provides a strong rationale for targeting DDR pathways in CSCs [ 26 ]. By selectively inhibiting these crucial repair mechanisms, it may be possible to overcome CSC-mediated resistance and achieve more complete tumor eradication, potentially preventing relapse (Table 2 ).

Table 2 Overview of major DNA damage response pathways and their roles in cancer Pathway Type of DNA damage repaired Key proteins involved Normal function Role in cancer cells/CSCs Refs. NER Bulky, helix-distorting lesions (UV, chemicals, cisplatin) XPA, XPC-RAD23B, XPG, RPA, ERCC1-XPF, TFIIH, PCNA, DNA polymerase δ/ε, DNA ligase I Replaces modified nucleotides with correct ones Dysregulation contributes to genomic instability; NER efficiency in CSCs may contribute to cisplatin resistance [ 6 ] BER Single-base damage (oxidation, alkylation, deamination), SSBs DNA glycosylases, APE1, PARP1, DNA polymerase β, XRCC1-DNA ligase α Replaces modified bases; repairs SSBs PARP1 hyperactivity in metabolically active cancer cells; contributes to drug resistance [ 6 ] MMR Base mismatches, small insertion/deletion loops from replication errors MutS, MutL, Exonucleases I, DNA polymerase δ Corrects replication errors and mismatches Defects lead to microsatellite instability (MSI), a hallmark of some cancers [ 6 ] HR DNA Double-Strand Breaks (DSBs) BRCA1, BRCA2, RAD51, MRN complex (RAD50, MRE11, NBS1) Error-free repair of DSBs using homologous template Often defective in cancer (e.g., BRCA mutations), creating synthetic lethal vulnerabilities; elevated HR activity in some CSCs (e.g., BCSCs, GSCs) contributes to radioresistance [ 6 ] NHEJ DNA Double-Strand Breaks (DSBs) DNA-PKcs, Ku70/80, XRCC4, Artemis, DNA Ligase Error-prone ligation of broken DNA ends Often hyperactive in cancer; elevated DNA-PKcs expression in GSCs contributes to radioresistance and stemness [ 6 ] Checkpoints Monitors DNA integrity, halts cell cycle ATM, ATR, Chk1, Chk2, p53, WEE1, Cyclin E, Myc, Ras Ensures DNA repair before cell cycle progression; induces apoptosis/senescence if damage is irreparable Dysregulation (e.g., p53 mutations) common in cancer; oncogene-induced replication stress increases reliance on ATR-Chk1; hyperactive checkpoints in CSCs prolong repair time, contributing to resistance [ 6 ]

The DDR paradox: how CSC resistance breeds vulnerability

To understand the therapeutic potential of synthetic lethality in this context, the relationship between CSC biology and DNA repair must be viewed as a chain of dependency:

The Stress Source: CSCs are driven by aggressive oncogenes (e.g., MYC, Ras) and metabolic changes that create chronic, high-level Replication Stress (RS) [ 56 , 64 ]. The Adaptation: To survive this self-inflicted stress, CSCs become ‘addicted’ to specific checkpoint kinases (like ATR and CHK1) and repair pathways (like Homologous Recombination) to prevent genomic collapse [ 27 , 56 , 65 ]. The Vulnerability: Unlike normal cells, which have low replication stress and backup repair options, CSCs have no ‘buffer’ [ 10 ]. The Strike: Synthetic lethality occurs when we block that specific adaptation (e.g., inhibiting ATR). The CSC cannot manage its inherent replication stress and dies, while normal cells survive [ 12 ].

The preceding sections establish a critical paradox: CSCs are defined by their enhanced DDR capacity, which confers profound resistance to chemotherapy and radiation [ 9 , 27 ]. However, this enhanced DDR is not a universal strengthening of all repair pathways. Instead, CSCs often suffer from high endogenous replication stress (due to oncogene activation) and a reliance on specific, error-prone repair mechanisms [ 64 ]. This forces them into a state of “DDR addiction”, where their survival becomes critically dependent on one or two hyperactive, compensatory pathways [ 66 ].

This dependency is the lynchpin that connects CSC resistance to synthetic lethal vulnerability. For example, while the enhanced G2/M checkpoint in glioblastoma stem cells (GSCs) [ 27 , 61 ] allows them to resist radiation, it also makes them uniquely dependent on the ATR-CHK1 pathway that governs this checkpoint. Inhibiting ATR in this context is not a general genotoxic insult; it is a precision strike that removes the single pillar supporting their genomic integrity, leading to catastrophic mitotic entry and cell death. Therefore, the goal of SL in CSCs is not just to “add more damage,” but to identify and exploit these specific, hyper-reliant pathways that CSCs have co-opted for their survival.

This reframes the central challenge from a descriptive one (what DDR pathways are active?) to an analytical one (which active pathways are compensatory vs. essential?). This raises key unresolved questions that this review will explore:

Is this “DDR addiction” a static, hard-wired feature of CSCs, or does it dynamically evolve in response to therapy and TME-induced stresses like hypoxia [ 18 ]? How do these dependencies differ across cancer types? For example, do GSCs (Ref 26) and breast CSCs [ 14 ] rely on the same checkpoints, or do their underlying oncogenic drivers create distinct SL vulnerabilities? Can we map these dependencies to identify novel, non-canonical SL partners beyond the well-established PARP and ATR/ATM axes?

Synthetic lethality: a precision oncology paradigm

Principles and biological rationale of synthetic lethality

Synthetic lethality (SL) is a genetic interaction where the simultaneous disruption of two genes or pathways leads to cell death, whereas the inactivation of either gene or pathway alone is compatible with cell viability [ 11 , 67 ] This concept provides a powerful framework for developing precision oncology strategies, as it allows for selective targeting of cancer cells that harbor specific genetic alterations, while sparing normal cells [ 12 ]. In cancer therapy, this often translates to pharmacologically inhibiting one gene product when the other is genetically inactivated in tumor cells [ 10 , 68 ]. This is particularly relevant for targets that are not classically “druggable,” such as loss-of-function mutations in tumor suppressor genes, as SL provides a means to exploit an indirect vulnerability [ 69 ].

The most celebrated example of SL in cancer is the interaction between BRCA1/2 mutations and Poly(ADP-ribose) polymerase (PARP) inhibition [ 14 ].

BRCA1/2 genes are crucial for homologous recombination (HR), an error-free DNA double-strand break (DSB) repair pathway [ 6 ]. PARP enzymes are involved in repairing single-strand breaks (SSBs) via the base excision repair (BER) pathway [ 6 , 70 ]. In BRCA -deficient cells, HR is compromised. Inhibiting PARP leads to an accumulation of unrepaired SSBs, which are converted into highly toxic DSBs during DNA replication. Since the primary DSB repair pathway (HR) is already defective in BRCA -mutant cancer cells, these cells cannot repair the accumulated DSBs and undergo cell death, while normal cells with intact BRCA function can repair the damage [ 10 ]. This selective toxicity has led to the clinical approval of PARP inhibitors for BRCA -mutated ovarian, breast, pancreatic, and prostate cancers [ 14 ].

Exploiting DDR vulnerabilities through synthetic lethality

The inherent dysregulation and hyperactivity of DDR pathways in cancer cells, particularly CSCs, create unique vulnerabilities that can be exploited using SL strategies. Cancer cells often exhibit defects in one DDR pathway, making them hyper-reliant on compensatory or alternative pathways for survival [ 71 ]. Inhibiting these compensatory pathways can induce SL.

ATR/ATM pathway dependencies ATM deficiency sensitizing to ATR inhibition: Cells with ATM deficiency are highly sensitive to ATR inhibition in preclinical models, a finding that has led to clinical trials of ATR inhibitors in ATM-deficient tumors [ 56 ]. This is because ATM and ATR have overlapping but distinct roles in DDR, and loss of ATM function increases reliance on ATR for replication stress response [ 56 , 65 ]. Oncogene-induced replication stress and ATRi: Many tumor cells, especially those with oncogene activation (e.g., Ras, Myc, Cyclin E), experience elevated replication stress and become critically dependent on the ATR pathway for survival. Inhibiting ATR in these contexts is selectively toxic [ 56 ]. This suggests that replication stress is a fundamental vulnerability that can be broadly exploited across many cancer types, regardless of specific BRCA mutations [ 72 ]. This broad applicability, coupled with the potential for synergy with conventional therapies, positions these inhibitors as crucial components of future cancer treatment regimens.

PARP inhibitor combinations Overcoming PARPi resistance: Combination therapies targeting cell cycle checkpoints (e.g., ATR-CHK1-WEE1) and PARPs can act synergistically to increase DNA errors and disrupt the cell cycle, overcoming PARP inhibitor resistance [ 73 ]. PARPi with ATRi: Combining PARP inhibitors with ATR inhibitors has shown success in preclinical models, particularly in PARPi-resistant and BRCA1/2 - and ATM -deficient ovarian and colon CSC models [ 74 ]. DNA-PKcs Inhibition: DNA-PKcs, a key NHEJ component, is often overexpressed in cancer and contributes to radioresistance [ 75 ]. Inhibiting DNA-PKcs can sensitize tumor cells to DNA-damaging agents, exploiting its role in safeguarding genomic integrity [ 75 ].

The expanding understanding of SL extends beyond the initial BRCA-PARP model, encompassing a diverse range of DDR defects and dependencies in various cancer types that can be therapeutically exploited [ 12 ]. The challenge lies in identifying reliable biomarkers for these new SL interactions [ 12 ].

Targeting CSCs via synthetic lethality and DDR inhibition

The enhanced DDR capacity and reliance on specific repair pathways make CSCs prime targets for synthetic lethal strategies. By selectively inhibiting these pathways, the intrinsic resistance of CSCs can be overcome, leading to their elimination.

PARP inhibitors: successes and resistance mechanisms in CSCs

The primary SL strategy here targets CSCs that are deficient in Homologous Recombination (HR) or those sustaining high levels of oxidative DNA damage; blocking PARP traps DNA lesions that the compromised CSC cannot repair [ 6 , 14 , 70 ].

PARP inhibitors (PARPi) are the first clinically approved drugs based on the synthetic lethality concept, showing significant efficacy in BRCA -mutated cancers [ 14 ].

Efficacy in BRCA-deficient CSCs: While CSCs are generally resistant to conventional treatments, including PARPi 64, in BRCA1 -mutant triple-negative breast cancers (TNBCs), CSCs can be sensitized to PARP inhibition by downregulating RAD51, a key HR protein [ 14 ] Human induced pluripotent stem cell (hiPSC)-based models of breast cancer with BRCA1/2 variants show selective sensitivity to Olaparib [ 76 ]. Preclinical studies in pediatric solid tumors: Olaparib has shown growth inhibition in bulk pediatric solid tumor cell lines, including neuroblastoma [ 77 ]. However, specific efficacy within the CSC subpopulation has been confirmed in BRCA1-mutant triple-negative breast cancer models, where PARP inhibition targeted the CD44+/CD24- fraction [ 14 ]. Resistance Mechanisms: Acquired resistance to PARPi is a significant challenge, with mechanisms including: Drug efflux: Upregulation of ABCB1 (MDR1) efflux pumps can lead to resistance, which can be reversed by ABCB1 inhibitors [ 78 ]. BRCA1/2 reversion mutations: Somatic reversion mutations can restore BRCA1/2 function, leading to PARPi resistance. Circulating cell-free DNA (cfDNA) analysis can help identify these mutations [ 78 ]. Homologous recombination repair (HRR) restoration: Any strengthening of the HRR process, such as loss of 53BP1, can confer PARPi resistance [ 78 ]. Bypass pathways: Activation of alternative oncogenic pathways like PI3K/Akt/mTOR and RAF/MEK can lead to PARPi resistance [ 8 ].

The dynamic nature of resistance to PARPi, where cancer cells, particularly CSCs, adapt to evade therapy, suggests that long-term efficacy will require continuous monitoring for resistance mechanisms (e.g., via liquid biopsy for BRCA reversion mutations 95) and the development of combinatorial strategies that pre-empt or overcome these adaptive responses. The challenge is not just to find a drug that works, but to anticipate and counteract the tumor’s evolutionary trajectory. While this highlights a clear vulnerability, the clinical reality is that acquired resistance to PARPi is common, often driven by mechanisms that restore HR function or increase drug efflux [ 78 ], necessitating the combination strategies discussed later in this review.

ATR and ATM inhibitors: exploiting replication stress and DDR defects in CSCs

ATR inhibition specifically targets the ‘Replication Stress’ vulnerability in CSCs. Because CSCs operate under high oncogenic drive, they rely heavily on ATR to stabilize replication forks; removing this support causes catastrophic fork collapse specifically in the stem cell population [ 27 , 56 ].

ATR and ATM kinases are master regulators of DDR, responding to replication stress and DNA double-strand breaks, respectively [ 6 ] Their inhibition selectively targets cancer cells with specific DDR defects or high replication stress.

ATR Inhibitors (ATRi): Mechanism: ATR responds to a wide range of DNA damage and replication problems, phosphorylating Chk1 to regulate cell cycle, replication fork stability, and DNA repair [ 56 ]. While ATRi toxicity is well-established in general cancer models, direct evidence in CSCs is observed in glioblastoma, where glioma stem cells (GSCs) exhibit heightened replication stress compared to differentiated tumor cells, rendering them acutely sensitive to ATR blockade [ 27 ]. Synthetic Lethality: ATRi exhibit synthetic lethality with ATM deficiency 52, ARID1A defects 76, and can synergize with PARP inhibitors [ 74 ]. Preclinical and Clinical Progress: ATRi (e.g., ceralasertib, tuvusertib, ART0380) are in various phases of clinical trials, both as monotherapy and in combination with chemotherapy, radiotherapy, or immunotherapy. They show promise in cancers like NSCLC and colorectal cancer [ 79 ]. ATM Inhibitors (ATMi): Mechanism: ATM is primarily activated by DNA double-strand breaks (DSBs) and orchestrates HR-mediated repair and cell cycle checkpoints [ 6 ]. Synthetic Lethality: ATMi can be effective in tumors with defects in other DDR nodes, exhibiting synthetic lethal interactions [ 80 ]. Preclinical and Clinical Progress: ATMi (e.g., AZD1390) are being tested in clinical trials, often in combination with radiation therapy for brain cancers like high-grade glioma [ 81 ] They also show potential to improve immunotherapy responses [ 80 ].

Oncogene activation is a common feature of cancer cells, and it directly induces replication stress [ 56 ]. This replication stress makes cancer cells, including CSCs, highly dependent on the ATR-CHK1 pathway for survival [ 56 ]. This implies that replication stress is not just a consequence of cancer but a fundamental vulnerability that can be broadly exploited. Targeting ATR-CHK1, WEE1, or other kinases involved in managing replication stress could be a universal strategy across many cancer types, regardless of specific.

BRCA mutations. This broad applicability, coupled with the potential for synergy with conventional therapies, positions these inhibitors as crucial components of future cancer treatment regimens. It must be noted that much of the CSC-specific data for these agents remains largely preclinical. A significant translational hurdle is their on-target/off-tumor toxicity to normal progenitors, particularly hematopoietic stem cells, which also rely on the ATR pathway for their own survival [ 82 ].

DNA-PKcs inhibitors and non-homologous end-joining in CSCs

This strategy targets quiescent (non-dividing) CSCs that rely on the Non-Homologous End Joining (NHEJ) pathway to repair double-strand breaks without entering the cell cycle [ 27 , 75 ].

DNA-PKcs is a key component of the non-homologous end-joining (NHEJ) pathway, responsible for repairing DNA double-strand breaks (DSBs) [ 6 ]. Its elevated expression and activity are correlated with cancer progression and resistance to treatment.

Role in Cancer and Resistance: DNA-PKcs expression is increased in various human cancers, including colorectal, esophageal, and nasopharyngeal carcinomas, and is associated with poor prognosis [ 75 ] Elevated DNA-PKcs is linked to radioresistance in cervical and prostate cancer cells [ 83 ]. Preclinical Studies: Inhibitors of DNA-PKcs (e.g., M3814) enhance the anti-tumor effect of chemotherapy (paclitaxel, etoposide) in NSCLC and have shown activity in ovarian cancer and AML [ 83 ]. They sensitize cancer cells to DSBs induced by chemo/radiotherapy [ 27 ]. Synthetic Lethality: DNA-PKcs inhibition can be synthetically lethal in certain contexts, for example, by blocking DSB repair and causing genomic instability [ 53 ] (Fig. 3 ). It is critical to note that while DNA-PKcs inhibitors are effective radiosensitizers in bulk tumors [ 27 , 83 ], evidence for their specific lethality in CSCs remains largely preclinical and inferred from the high expression of DNA-PKcs in Glioblastoma Stem Cells (GSCs).

Fig. 3 Targeting DNA damage response (DDR) pathways in cancer stem cells (CSCs). ATR inhibitors (ATRi) disrupt ATR–CHK1 signaling, exploiting replication stress dependence in CSCs. ATM inhibitors (ATMi) block ATM-mediated homologous recombination (HR) repair of DNA double-strand breaks. DNA-PKcs inhibitors impair the non-homologous end-joining (NHEJ) pathway by targeting DNA-dependent protein kinase catalytic subunit (DNA-PKcs). Together, these inhibitors exploit replication stress and DDR defects, offering synthetic lethality and enhanced sensitivity to chemo- and radiotherapy (Images were drawn by AI and further edited with adobe illustrator)

Translational hurdles: resistance, toxicity, and the biomarker gap

Despite the compelling preclinical rationale, the translation of DDR inhibitors for CSC targeting faces three significant and unresolved hurdles that are a major focus of current research.

Acquired Resistance: The central challenge in SL is its evolutionary fragility. Tumors are highly plastic and develop resistance to DDR inhibitors through numerous mechanisms. In the context of PARPi, this famously includes somatic reversion mutations in BRCA1/2 that restore a functional HR pathway [ 78 ]. However, more complex, non-genetic mechanisms are emerging, such as the loss of 53BP1 (which restores HR in BRCA1 -mutant cells) or the activation of parallel repair pathways [ 84 ]. Critically, many CSCs can also leverage their innate resistance mechanisms, such as the upregulation of ABCB1 drug efflux pumps, to simply expel the inhibitor, a challenge that plagues many targeted therapies [ 78 ]. On-Target/Off-Tumor Toxicity: The assumption that normal, healthy cells will be spared is overly simplistic. Many DDR inhibitors, particularly ATR and CHK1 inhibitors, exhibit significant hematological toxicity (e.g., thrombocytopenia, neutropenia) [ 82 ]. This is because normal hematopoietic stem and progenitor cells are also highly proliferative and rely on the ATR pathway to manage their own baseline replication stress. This narrow therapeutic window is a critical barrier to achieving effective dosing and is a major reason for failure in clinical trials, complicating their use in combination regimens. The Biomarker Gap: The success of PARPi was built on a clear predictive biomarker: BRCA1/2 mutation. For nearly all other DDR inhibitors, no such robust biomarker exists. For example, identifying which patients will respond to an ATR inhibitor is a major gap. While ATM mutations are a rational SL partner [ 56 ], they are not as prevalent. Current research is urgently focused on developing pharmacodynamic (PD) biomarkers (e.g., measuring p-CHK1 or γ-H2AX in tumor or surrogate tissues) to prove target engagement, and functional biomarkers (e.g., RAD51 foci assays) to assess HR competency, rather than relying on genetic mutations alone [ 85 ]. These complex challenges of resistance, toxicity, and biomarker identification are not just theoretical; they are the primary focus of current clinical investigation. Table 3 provides an illustrative summary of selected clinical trials that are attempting to address these very issues, often by testing novel combinations, using advanced trial designs, or targeting specific, CSC-rich tumors like glioblastoma.

Table 3 Selected clinical trials investigating DDR inhibitors with high relevance to CSCs and SL Inhibitor class Drug example(s) Target(s) Example trial ID Rationale/unresolved question (biomarker) Evidence for CSC targeting PARPi + Immunotherapy Talazoparib + Avelumab PARP1/2 + PD-L1 NCT03642132 DDR & Immunotherapy: Testing if PARP inhibition can trigger cGAS-STING and enhance response to immune checkpoint blockade Inferred: Aims to reverse TME immunosuppression, a key CSC defense mechanism PARPi + Chemotherapy Olaparib + Temozolomide PARP1/2 NCT05633381 Sensitizing GSCs: Specifically testing if PARPi can sensitize recurrent glioblastoma (a highly CSC-rich tumor) to chemotherapy (TMZ) High: Trial rationale specifically targets glioblastoma, a tumor defined by a high frequency of therapy-resistant GSCs. ATRi + Immunotherapy Ceralasertib + Durvalumab ATR + PD-L1 NCT05450692 Targeting RS & Immunity: Exploiting high replication stress (ATRi) while simultaneously blocking the PD-L1 immune checkpoint Inferred: Targets replication stress, a known CSC vulnerability (NEW REF [ 1 ]) ATMi + Radiation AZD1390 + Radiation ATM NCT03423628 Radiosensitization: AZD1390 is designed to cross the blood-brain barrier. This trial directly tests sensitization of glioblastoma (a CSC-rich tumor) to radiation High: Trial specifically targets glioblastoma; GSCs are the key driver of radioresistance WEE1i + PARPi Adavosertib + Olaparib WEE1 + PARP NCT03579316 Overcoming PARPi Resistance: Testing if targeting the G2/M checkpoint (WEE1) can re-sensitize BRCA -mutant tumors that have developed PARPi resistance [ 86 ] Inferred: Targets acquired resistance, a process often driven by CSC plasticity and survival

Emerging synthetic lethal targets and combinatorial strategies

The field of synthetic lethality is continuously expanding beyond the canonical DDR pathways, with new targets and combinations emerging (Table 3 ).

Methylation Synthetic Lethality: Aberrant epigenetic changes, particularly DNA methylation, are more frequent than gene mutations in some cancers. Targeting these methylation patterns can lead to synthetic lethal interactions involving DNA damage repair genes, metabolic pathway genes, and paralogs [ 10 ]. Other DDR-related targets: Beyond ATM, ATR, PARP, and DNA-PKcs, other DDR proteins like WRN, RAD51, Sam68, and cell cycle checkpoint regulators (CHK1/WEE1, Aurora A/B, TPX2) are being investigated for synthetic lethal interactions [ 53 ]. Metabolic pathway genes: Targeting metabolic vulnerabilities, such as those related to nucleotide synthesis (e.g., PRPS2, IMPDH1/2, CTPS1), can also create synthetic lethal opportunities in MYC-driven cancers [ 53 ]. Combination Therapies: The most promising strategies involve combining DDR inhibitors with conventional therapies (chemotherapy, radiation) or other targeted agents, including immune checkpoint inhibitors [ 11 ]. This aims to enhance efficacy, overcome resistance, and improve patient survival (Table 4 ). While the biological rationale for these combinations is strong, clinical data demonstrating synergistic CSC eradication is still lacking, and the potential for overlapping toxicities remains a significant and unresolved challenge.

Table 4 Selected DDR inhibitors in preclinical and clinical development for cancer Inhibitor class Drug examples Target(s) Mechanism of action Preclinical progress (CSC-specific if available) Clinical trial status and indications Refs. PARP Inhibitors Olaparib, Niraparib, Talazoparib, Rucaparib PARP1, PARP2 Inhibits SSB repair, traps PARP on DNA, leads to DSBs in HR-deficient cells (synthetic lethality) Sensitizes BRCA1 -mutant TNBC CSCs (RAD51 downregulation); growth inhibition in pediatric solid tumor cell lines (neuroblastoma, Ewing sarcoma) 64 Approved for BRCA -mutmutated ovarian, breast, pancreatic, prostate cancers; ongoing trials for resistance, combinations [ 14 ] ATR Inhibitors Ceralasertib, Tuvusertib, ART0380, VE-821, NU6027 ATR, Chk1 Inhibits ATR-Chk1 pathway, disrupts replication fork stability, causes genomic instability, selectively toxic in cells with high oncogene-induced replication stress Sensitizes ATM-deficient cells; effective in cells with ARID1A defects; synergizes with PARPi; radiosensitizes glioma CSCs Phase 1, 2, 3 trials (monotherapy, combinations with chemo, RT, ICI) for NSCLC, colorectal, solid tumors with ATM alterations [ 80 ] [ 12 ] ATM Inhibitors AZD1390, M3814 (DNA-PK/ATM dual) ATM, Chk1/2, p53 Inhibits ATM, disrupts DSB repair (HR), cell cycle checkpoints, promotes genomic instability Radiosensitizes adult and pediatric glioblastoma tumors; effective in tumors with other DDR defects Clinical trials (combinations with RT, other DDRi) for high-grade glioma, solid tumors with ATM alterations [ 56 ] [ 12 ] DNA-PKcs Inhibitors M3814, Nedisertib, AZD7648, M9831 (VX-984) DNA-PKcs Inhibits NHEJ pathway, blocks DSB repair, causes genomic instability Reduces GSC stemness, sensitizes glioblastoma xenografts to RT; enhances chemo effects in NSCLC, ovarian, AML 58 Preclinical to early phase clinical trials (combinations with chemo, RT) [ 79 ]

Synergistic platforms: combining advanced modalities with DDR/SL strategies

While direct inhibition of DDR pathways is a core strategy, its efficacy can be dramatically enhanced by combining it with advanced therapeutic platforms. These modalities are not just “adjacent approaches”; they are critical tools to solve the primary challenges of DDRi, such as drug delivery, target specificity, and overcoming the immunosuppressive TME. This section analyzes how these platforms can be integrated with a DDR/SL-based approach to specifically eradicate CSCs.

Small molecule inhibitors (SMIs) of stemness pathways

Instead of targeting DDR pathways directly, SMIs targeting core stemness pathways (e.g., Wnt, Notch, Hedgehog) can be a powerful synergistic partner for DDRi. CSCs are often addicted to these pathways for self-renewal, but this hyper-proliferative signaling is also a primary source of oncogene-induced replication stress [ 64 ]. By inhibiting these pathways, we may not only halt self-renewal but also create new DDR dependencies, potentially sensitizing CSCs to ATR or WEE1 inhibitors.

Antibody-drug conjugates (ADCs) as DDRi delivery systems

The challenge of systemic toxicity (e.g., hematological toxicity from ATRi) can be overcome by improving targeted delivery. ADCs are a platform to achieve this. By arming a CSC-specific antibody (e.g., targeting MUC1, EpCAM, or CD44v6) with a highly potent DDR inhibitor payload (e.g., a PARPi or CHK1i), this approach can deliver the drug directly to the CSC population. This emerging “ADC-DDRi” class could achieve a far wider therapeutic window and overcome resistance driven by systemic drug efflux [ 87 , 88 ].

Nanoparticles for co-delivery and overcoming resistance

Similarly, nanoparticle-based systems are ideal for solving key DDRi challenges in CSCs. Their primary advantage is the ability to co-encapsulate multiple agents (e.g., a traditional cytotoxic drug and a PARP inhibitor) within a single particle. This ensures the drugs arrive at the CSC simultaneously, maximizing synergistic killing. Furthermore, NPs can be engineered to bypass ABC transporters, a key CSC resistance mechanism [ 89 ], and can be targeted to the acidic or hypoxic CSC niche [ 90 ].

Integrating CAR cell therapies with DDRi-driven immunomodulation

The limitation of CAR-T cell therapy in solid tumors is the immunosuppressive TME, which CSCs actively maintain (Your Ref: [ 23 , 91 ]). A highly promising and relevant strategy is to combine CAR-T with DDR inhibitors. As discussed in Sect. 4.4, DDR inhibitors (especially PARPi and ATRi) can induce cytoplasmic micronuclei, activating the cGAS-STING pathway and triggering a robust Type I interferon response. This “inflames” the tumor microenvironment, increasing T-cell infiltration and making CSCs more “visible” to both the innate immune system and the engineered CAR-T cells [ 91 ].

Other targeted approaches

Other modalities, such as glycan-based therapies, are also in development. These aim to target the aberrant glycosylation on the CSC surface (e.g., TACAs like Tn and sLeX) [ 92 ], which are involved in immune evasion and metastasis. While less directly linked to DDR, they represent a parallel strategy for targeting CSC-specific biology.

Challenges and future directions

Challenges in CSC targeting

Targeting CSCs effectively faces several significant hurdles. The foremost challenges are their biological elusiveness and adaptive nature. These include:

Heterogeneity and Plasticity: CSC populations are highly heterogeneous and can dynamically switch between stem-like and differentiated states (phenotypic plasticity), making consistent identification and targeting difficult [ 1 , 18 ]. Lack of Specific Markers: Many CSC markers (e.g., CD44, CD133) are also expressed on normal adult stem cells, creating a high risk of on-target/off-tumor toxicities (Your Ref: [ 93 ]). The Integrated Resistance Network: As detailed in Sect. 2.2, CSCs are protected by a complex and interconnected network of intrinsic and extrinsic factors, including quiescence, high drug efflux, metabolic plasticity, and a deeply immunosuppressive TME, which must be overcome simultaneously.

Challenges in DDR targeting and synthetic lethality

Despite the promise of DDR targeting and synthetic lethality, several challenges persist:

Acquired Resistance: Tumors often develop resistance to DDR inhibitors through various mechanisms, including drug efflux, restoration of DNA repair pathways (e.g., BRCA reversion mutations), and activation of bypass signaling pathways [ 8 ]. Specificity and Toxicity: Achieving high specificity for cancer cells while minimizing toxicity to normal tissues, which also rely on DDR pathways, remains a hurdle [ 56 ]. Biomarker Identification: The identification of reliable predictive and pharmacodynamic biomarkers is crucial for patient selection and monitoring response to DDR inhibitors, especially for novel synthetic lethal interactions [ 12 ]. Tumor Heterogeneity: The genomic and phenotypic heterogeneity within and between tumors complicates the application of DDR-targeted therapies, as different subclones may have varying DDR defects or compensatory mechanisms [ 93 ].

Challenges in advanced therapeutic modalities

CAR Cell Therapies: Solid Tumor Barriers: Poor tumor infiltration, T cell exhaustion, and the immunosuppressive TME limit efficacy in solid tumors [ 94 ]. On-target/Off-tumor Toxicity: Targeting antigens shared with normal cells can lead to severe side effects (e.g., cytokine release syndrome, neurotoxicity, B cell aplasia) 296. Antigen Escape: Tumors can downregulate or lose target antigen expression, leading to relapse [ 95 ]. Manufacturing and Cost: The complex, time-consuming, and costly manufacturing process limits accessibility and scalability [ 96 ]. Persistence: Limited in vivo persistence of CAR-T and CAR-NK cells can lead to tumor recurrence [ 35 ]. Nanoparticle Delivery: Challenges include optimizing drug loading, controlled release, stability, and penetration into complex tumor tissues [ 89 ].

The promise of integrated diagnostic platforms for precision oncology

To overcome the complexities of CSC heterogeneity, dynamic resistance mechanisms, and the challenges in therapeutic translation, integrated diagnostic platforms are emerging as a transformative approach in precision oncology.

Multi-omics Profiling: Integrating genomic, epigenomic, transcriptomic, proteomic, and metabolomic data from patient tumors provides a comprehensive understanding of cancer biology. However, to move beyond these general platforms, physicians require concrete predictive biomarkers that link a specific DDR defect to the appropriate synthetic lethal drug [ 14 , 78 , 92 , 97 , 98 ]. While some markers (like BRCA mutations) are standard of care, others are emerging as critical indicators of CSC vulnerability (Table 5 ).

Table 5 Actionable biomarkers for targeting CSCs via synthetic lethality Biomarker Defect Indicated Suggested SL Therapy Level of CSC Evidence Refs. BRCA1/2 Mutation Loss of Homologous Recombination (HR) PARP Inhibitor Strong: Confirmed in Breast/Ovarian CSCs (RAD51 low) [ 14 , 76 ] ATM Loss Loss of DSB repair signaling ATR Inhibitor Moderate: Strong in bulk tumor; inferred in CSCs via synthetic lethality principles [ 56 , 80 ] CCNE1 Amplification/High MYC High Replication Stress ATR or WEE1 Inhibitor Strong: High oncogene expression drives CSC replication stress addiction [ 53 , 92 ] DNA-PKcs Overexpression Hyperactive NHEJ Repair DNA-PK Inhibitor + Radiation Strong: High levels in Glioblastoma Stem Cells correlate with radioresistance [ 27 , 75 ] ARID1A Mutation Chromatin remodeling defect ATR Inhibitor Emerging: Preclinical evidence suggests synthetic lethality in gynecologic cancers [ 12 ] High Microsatellite Instability (MSI-H) Mismatch Repair Deficiency Immunotherapy + DDRi Strong: High mutational burden makes CSCs visible to immune system [ 6 , 51 ]

Clinically, this means that profiling a patient’s tumor for ‘Replication Stress Signatures’ (e.g., CCNE1 or MYC amplification) rather than just standard mutations could identify those who will benefit from ATR inhibitors to deplete the resistant stem cell reservoir. Integrated platforms are the essential tools to identify these markers in the correct CSC subpopulation: liquid biopsy can detect cfDNA-based BRCA reversion mutations, while single-cell profiling can confirm that a specific DDR defect exists within the rare CSC clone, not just the bulk tumor [ 78 , 97 ].

Liquid Biopsy: Non-invasive liquid biopsies (analyzing circulating tumor cells (CTCs), cell-free DNA (cfDNA), extracellular vesicles (EVs)) enable real-time monitoring of tumor evolution, early relapse detection, and identification of actionable targets, overcoming limitations of tissue biopsies (e.g., sampling errors, intratumor heterogeneity). Single-Cell Profiling: Single-cell RNA sequencing (scRNA-seq) and ATAC-seq provide unprecedented resolution to characterize cellular heterogeneity, identify distinct tumor cell subpopulations, and understand TME rewiring during therapy, revealing intrinsic and extrinsic regulators of therapy response [ 97 ]. Artificial Intelligence (AI) and Machine Learning (ML): AI/ML algorithms integrate diverse data types (imaging, molecular, clinical, single-cell) to improve diagnostic accuracy, predict prognosis, classify cancer subtypes, identify therapeutic targets, and accelerate clinical decision-making.

These integrated platforms hold the potential to revolutionize precision oncology by enabling earlier detection, more accurate risk stratification, personalized treatment selection, and real-time monitoring of response, ultimately improving patient outcomes and quality of life.

Conclusions

The landscape of cancer therapy is profoundly shaped by the intricate biology of Cancer Stem Cells (CSCs) and their remarkable capacity for self-renewal, metastasis, and, critically, therapeutic resistance. This resistance is frequently rooted in their highly efficient DNA Damage Response (DDR) mechanisms, metabolic plasticity, and ability to sculpt an immunosuppressive tumor microenvironment. The comprehensive analysis presented herein underscores that these resistance mechanisms are not isolated but rather form a dynamic and interconnected network, presenting a significant hurdle for conventional monotherapies.

The genetic concept of Synthetic Lethality (SL) offers a powerful and selective paradigm to overcome CSC-mediated resistance. The clinical success of PARP inhibitors in BRCA -deficient cancers serves as a compelling proof-of-principle, demonstrating that exploiting a specific DDR defect by inhibiting a compensatory pathway can lead to selective cancer cell death. The expanding exploration of SL beyond BRCA -PARP interactions, particularly involving ATR, ATM, and DNA-PKcs inhibitors, highlights a growing recognition of replication stress and other DDR dependencies as broad vulnerabilities across diverse cancer types. However, the adaptive nature of cancer cells, which can develop resistance to DDR inhibitors through various bypass pathways or functional restorations, necessitates a continuous evolution of therapeutic strategies.

Advanced therapeutic modalities, including novel small molecule inhibitors, highly targeted antibody-drug conjugates (ADCs), and engineered CAR cell therapies (CAR-T, CAR-NK, CAR-NKT), are being developed to specifically eliminate CSCs. These approaches aim to leverage CSC-specific surface markers, disrupt their aberrant signaling and metabolic pathways, and overcome the immunosuppressive tumor microenvironment. While CAR therapies have revolutionized hematologic malignancies, their application in solid tumors remains challenging due to issues like tumor heterogeneity, antigen escape, and on-target/off-tumor toxicities. Nanoparticle-based drug delivery systems are emerging as crucial tools to enhance the specificity and efficacy of these agents by improving drug delivery to CSCs and overcoming efflux mechanisms.

Ultimately, achieving durable cancer control requires a multi-pronged approach that integrates the latest biological insights with advanced technological platforms. The advent of integrated diagnostic platforms, leveraging AI, liquid biopsy, multi-omics, and single-cell profiling, is poised to transform precision oncology. These platforms enable unprecedented resolution in understanding tumor heterogeneity, real-time monitoring of disease progression and resistance, and the identification of patient-specific vulnerabilities. By combining these diagnostic capabilities with innovative therapeutic strategies that exploit synthetic lethality and target CSCs, the field is moving towards a future where cancer treatment is truly personalized, adaptive, and capable of achieving more profound and lasting responses for patients. Continued investment in understanding the complex interplay between CSC biology, DDR, and the tumor microenvironment, coupled with the development of robust biomarkers and combinatorial therapies, will be critical to realizing the full potential of this precision oncology revolution.

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Acknowledgements

Figures were generated from adapted figures provided by Servier Medical Art (Servier; https://smart.servier.com/ ), licensed under a Creative Commons Attribution 4.0 Unported License. We also acknowledge Canva Pty Ltd (Sydney, Australia) for supplying design software that facilitated the creation of figures; all graphical content remains the original work of the authors. During the preparation of this work, the authors used Grok and ChatGPT in order to improve the writing process and to enhance the readability and language of the manuscript. After using this tool/service, the authors reviewed and edited the content as needed and take full responsibility for the content of the published article.

Author contributions

Usamah Sayed, Waleed K. Abdulsahib, Ihsan Khudhair Jasim, H. Malathi, Priya Priyadarshini Nayak, D. Alex Anand, Gunjan Mukherjee, Aashna Sinha, and Sadridin Eshkaraev contributed to the conception, design, and drafting of the manuscript. All authors read and approved the final version of the manuscript.

Funding

Not applicable.

Data availability

No datasets were generated or analysed during the current study.

Declarations

Ethical approval and consent to participate

Not applicable.

Consent for publication

Not applicable.

Competing interests

The authors declare no competing interests.

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