HR is a multistep process that prevents genomic instability and maintains cellular homeostasis. After DSBs occur, BRCA1 promotes HR pathways. First, the MRN complex (mre11-rad50-nbs1) activates this pathway by coupling to DSB (Belan et al., 2022). The MRN complex acts synergistically with BRCA1 and CtIP endonucleases to mediate DNA end resection (Kishkevich et al., 2022). Furthermore, the MRN complex triggers ATM, which in turn initiates PALB2, BRCA1, and BRCA2 expressions (Murciano-Goroff et al., 2022; Peng et al., 2023). Subsequently, RAD51 loads onto the DNA damage site to form a nucleoprotein, which further invades sister chromosomes to search for orthologous DNA sequences that can be used as templates for novel DNA synthesis (Cleary et al., 2020; Murciano-Goroff et al., 2022).

By losing ATM in cancer cells, HR is undermined; therefore, when DNA is damaged, these cancer cells depend on the rest of the DDR pathway for repair (Kaminski et al., 2022). Cancers with specific HR defects can be treated with targeted agents that inhibit HR proteins.

In cancer cells, a mechanism of telomere lengthening via homologous targeted repair (i.e., ALT) is similar to HR and triggers DNA repair to maintain telomere length (Kaminski et al., 2022). TERRA is a transcription factor involved in telomere elongation. Chia et al. reported that several HR-related proteins, including RAD50, BRCA1, WRN, ATR, and WRNIP1, are potential TERRA-interacting proteins (Guh et al., 2022). TERRA initiates RAD51-dependent strand invasion (Feretzaki et al., 2020), whereas BRCA1 binds to and represses TERRA transcription (Vohhodina et al., 2021). A strong correlation exists between telomere function and the DNA damage response, particularly HR, which might be a potential therapeutic target for ALT cancer treatment.

NHEJ is a DDR pathway for the repair of DSBs and is activated by 53BP1, RIF1, and the shieldin complex. NHEJ includes classical non-homologous end-joining (cNHEJ), alternative non-homologous end-joining (alt-EJ), and MMEJ (Gómez-Cabello et al., 2022). cNHEJ utilizes nonspecific ligation to correct DNA breaks, resulting in error-prone repair that can occur at any time in the cell cycle (van de Kooij et al., 2022). cNHEJ is initiated via binding of the Ku70–Ku80 (also known as XRCC6–XRCC5) heterodimer to DSB ends (Luedeman et al., 2022). This event activates DNA-PK, which, in turn, activates a multi-protein complex of XRCC4, Artemis, and DNA ligase. Although the NHEJ mechanism is simpler than the HR mechanism, it can sometimes result in rearrangements, whereas the HR mechanism does not produce errors (Findlay et al., 2018). Alt-EJ primarily utilizes microhomologous fragments (2–25 bp) of the damaged region to generate an annealing reaction and remove nonhomologous ssDNA (Oanh et al., 2022). Alt-EJ is poly ADP-ribose polymerase 1 (PARP1)-dependent, and polymerase θ (Polθ) mediates break repair after MRN complex and PARP1-binding double-strand breaks. When HR is defective in tumors, it causes an enhanced reliance on alt-EJ. The correlation between the effect of alt-EJ on Polθ indicates that Polθ inhibitors are likely to be potent in HR-deficient tumors (Findlay et al., 2018; Scully et al., 2019; Shibata and Jeggo, 2020; Murciano-Goroff et al., 2022).

Ribes-Zamora et al. have determined the role of shelterin in suppressing the NHEJ function of Ku in human telomeres (Ribes-Zamora et al., 2013). Telomere fusion occurs when NHEJ acts on uncapped telomeres (Ueno, 2023). As the center of telomere maintenance and structure, the complex between TRF2 and Rap1 blocks NHEJ, and together with DNA-PK, inhibits telomere end-joining (Arat and Griffith, 2012). NHP2 is a component of the telomerase–holoenzyme complex. In telomere RNA subunit (hTR)-expressing ALT + cells, NHP2 is downregulated, and 53BP1 foci at telomeres are increased. The depletion of NHP2 in hTR-expressing cells rarely reduces the total 53BP1 level, but does decrease TIF reduction compared to NHP2 depletion in non-hTR-expressing cells (Raghunandan et al., 2021). Therefore, NHEJ factors are attractive targets for cancer therapies because of the reliance of tumor cell division on DNA repair mechanisms.

BIR primarily repairs single-ended DSBs similar to those resulting from telomere encroachment or replication fork crashes (Kockler et al., 2021). BIR was first reported in bacteriophage T4 and occurs in both mammalian cells and humans (Kreuzer, 2000). One study found that the overexpression of oncogenes activates BIR in human cells, resulting in chromosomal rearrangements (Elango et al., 2019). In ALT + cells, BIR is active during the G2 stage of the cell cycle and RAD52 is recruited to the replication stress site, a process that requires the two regulatory subunits of DNA polymerase δ, namely, POLD3 and POLD4 (Silva et al., 2019). ALT is a BIR that functions through both RAD52-and non-dependent processes. (Verma et al., 2019). In ALT cells, RAD52 is primarily responsible for D-loop formation and mediates the RAD52-dependent BIR process, whereas RAD51AP1 is primarily responsible for TERRA-mediated R-loop formation in telomeres, which promotes G4 formation. Subsequently, G4 promotes R-loop-to-D-loop conversion, which promotes ALT (Kaminski et al., 2022; Yadav et al., 2022). Moreover, BIR can trigger the SUMOylation of PIAS4-mediated TRF2, and the deprivation of PIAS4 renders APB devoid of repair proteins, which in turn compromises the synthesis of ALT telomeres (Zhang et al., 2021). Therefore, understanding the role of BIR in the treatment of ALT cancer cells is vital (Elango et al., 2017).

SSA is a double-stranded oligonucleotide with a 3′ overhang of three random nucleotides that can be efficiently ligated to the 3’ end of single-stranded DNA using T4 DNA ligase (Wu et al., 2018). Similar to the alt-EJ mechanism, SSA requires homologous DNA sites to catalyze DSB repair. However, SSA can occur over long stretches of DNA and result in large deletions that can cause intrachromosomal translocations. Mechanistically, SSA is inhibited by RAD51. Unlike alt-EJ, which requires PARP and Polθ, SSA requires RAD52 to influence the annealing of the homologous stretches of ssDNA (Scully et al., 2019; Vancevska et al., 2020; Subecz et al., 2021).

The mammalian ERCC1/XPF endonuclease plays an important role in DSB repair via SSA (Kim et al., 2020). TERRA triggers XPF localization to telomeres and results in FANCM deficiency, eventually leading to DSBs (Guh et al., 2022). SLX4, a coordinator of multiple DNA structure-specific endonucleases, plays important roles in several DNA repair pathways. The Slx4-Rad1 complex is required for the SSA pathway, in which the Mec1/Tel1-dependent phosphorylation of Slx4 is essential (Saito et al., 2009). SLX4 cooperates with XPF for interstrand DNA crosslink repair and is required for XPF-mediated DDR at ALT telomeres (Guh et al., 2022). Therefore, therapies targeting the SSA repair pathway may be useful for treating ALT cancer cells (Pfitzer et al., 2019).

BER is initiated by an impaired base and is substituted by de novo-synthesized DNA. Then, APE (depurine/depyrimidine nuclease) cleaves it to form a 3′OH end at the site of damage (Szymanski et al., 2022; Tang et al., 2022). Finally, DNA ligase and polymerase are used to bridge these gaps (Farag et al., 2022). The NER mechanism involves the removal of damaged DNA by the excision repair cross-complementary protein 1 (ERCCI), which is substituted with normal DNA replication (Elango et al., 2017; Scully et al., 2019). Defects in BER are correlated with premature aging, and BER genes are overexpressed in various cancers, such as POLβ, XRCC1, and APE1, thus indicating that BER is essential for genome maintenance (Somuncu et al., 2020).

XPF is an NER factor with nucleic acid endonuclease activity and is the most enriched TERRA-binding protein according to mass spectrum results (Guh et al., 2022). XPF can generate DSBs while promoting DDR in ALT telomeres (Guh et al., 2022). Yang et al. discovered that the principal mechanism of telomere replication may be linked to TFIIH, because as an NER element, TFIIH is an important factor in TRF1 and its absence results in several telomere replication phenotypes (Yang et al., 2022).

MMR restores the nucleotide sequence in DNA molecules with mismatched bases. When microsatellite instability (MSI) occurs in an organism, MMR proteins repair the errors. Normal functioning of the MMR protein repairs the MSI and maintains microsatellite stability; however, when the MMR protein is absent, MSI is not repaired, and it will gradually accumulate, thus resulting in high MSI. MSI is typically found in colorectal cancer but can also occur in gastric and endometrial cancers (Scully et al., 2019; Shibata and Jeggo, 2020; Ngo et al., 2021).

Recent literature suggests that the loss of MMR function may play a significant role in ALT activity in human cancer cell lines; however, this correlation has not been confirmed in human primary tumors. Furthermore, the loss of MMR function is associated with ALT, improved organism survival, and health in yeast and mice, thus supporting the role of the loss of the MMR pathway in promoting the development of ALT (Stundon et al., 2022). MMR is initiated by one of two heterodimers: MSH2/MSH6 (MutSα) and MSH2/MSH3 (MutSβ). MutSα binds to base–base mismatches or the insertion and deletion loops of 1–3 nt, whereas MutSβ binds to insertion and deletion loops containing up to 16 nt (Kunkel and Erie, 2015). A recent study revealed that MutSα restricts telomere extension via ALT-associated homology-directed repair in human cancer cells (Barroso-González et al., 2021). MutSβ precludes the aggregation of R-loops and telomeric G-quadruplex (G4) structures (Sakellariou et al., 2022). Additionally, SLX4 is associated with the proteins MSH2-MSH1 and TRF2 (Ouyang et al., 2015). These associations suggest a link between telomeres and MMR, thus offering potential therapeutic alternatives for cancer treatment.

Owing to the prevalence of telomerase-positive cancer in all cancer patients, targeted telomerase therapy is considered a potential approach for cancer treatment. Several promising candidates are currently being investigated in clinical trials and pre-clinical studies (Table 2).

Small-molecule telomerase reverse transcriptase (TERT) has been well studied and has achieved good practical prospects. BIBR1532 is a representative drug of this type that binds to TERT at the non-catalytic site and inhibits telomerase activity in a non-competitive manner (Liu B. et al., 2022). Its cytotoxicity is primarily caused by direct damage to the telomere structure, resulting in the loss of TRF2 binding, which induces telomere dysfunction, acts as a telomere end-to-end fusion, and increases p53 activation. The preclinical studies have shown that BIBR1532 is effective against several cancer cell lines, including breast cancer, fibrosarcoma, endometrial cancer, and leukemia (Qin and Guo, 2022; Al-Karmalawy et al., 2023). Due to the relative instability of epigallocatechin gallate (EGCG), improved TERT inhibitors synthesized from EGCG-related fractions have been developed (Wu et al., 2020). Among these, MST-312 has been confirmed to be involved in various types of cancers. In breast, lung, and colon cancers, MST-312 treatment significantly downregulates TERT expression, reduces telomerase activity, and results in telomere shortening. It can also lead to cell cycle arrest and apoptosis in cancer cells (Wu et al., 2020). However, MST-312 works only on cancer cells with short telomeres, according to the time required for telomere shortening to a critical length (90 d) (Fernandes et al., 2022). Nu-1 and erythromycin antibiotics are other TERT inhibitors that can directly bind to the TERT catalytic domain and block TERT transcription. However, these drugs have not been well studied and have only been investigated in early clinical research because of their low potency and slow onset of cytotoxicity (Ameri et al., 2019; Wu et al., 2020). G4 ligands inhibit telomerase binding to telomeric DNA, eventually inhibiting telomerase activity (Tiek et al., 2022). G4-stabilizing ligands include telomeric repressors, TMPyP4, RHPS4, pyridostatin and telomestatin (Teng et al., 2021; Alessandrini et al., 2022; Liu LY. et al., 2022; Iida et al., 2022). Owing to the high levels of G-rich DNA throughout the genome, particularly in the promoter regions of oncogenes, G4 ligands pose numerous risks when used as telomerase inhibitors and might have a significant off-target effect (Yan et al., 2021; Shankar et al., 2022; Tsai et al., 2022). Oligonucleotides can form stable double-stranded bodies with complementary DNA, thereby disrupting hTR function. Binding to the hTR sequence template effectively inhibits the catalytic action of telomeric repeat addition, thereby inhibiting telomerase activity. The representative drug in this category is imetelstat (GRN163L), which has been tested in different tumor models and is currently the only anti-telomerase oligonucleotide in clinical use (Djojosubroto et al., 2005; Fischer-Mertens et al., 2022).

Regarding ALT-positive cancer cells, the development of cancer drugs targeting ALT can be traced back to its upstream and downstream pathways, particularly the DDR. In ALT-positive cancer cells, PARP inhibitors can cause TRF2 to dissociate from telomeres, thereby stimulating the inappropriate repair of non-homologous end connections. ATM and ATR inhibitors can also be used for the treatment of ALT-positive cancers. AZD0156, an ATM inhibitor, has been used to treat ALT neuroblastomas and overcomes chemotherapy resistance (Koneru et al., 2021). As an inhibitor of the FANCM-BTR interaction (Wu et al., 2023), PIP-199 may be selectively toxic to ALT cancer cells (Lu et al., 2019), rendering it a potential therapeutic target. Tetra-Pt (bpy), a cisplatin derivative that targets telomeric G-quadruplexes, severely inhibits the growth of ALT cell xenograft tumors, indicating that it may be a novel oncotherapeutic agent for targeting ALT cancer cells (Zheng et al., 2017). In addition, bpy disrupts telomere maintenance in telomerase cancer cells, further elucidating the function of G-quadruplexes in the human genome (Shen et al., 2022). The potential of bpy as a chemotherapeutic target has been demonstrated in both ALT-positive and telomerase cancer cells.

The shelterin protein complex prevents chromosome ends from being recognised as DSB and activates the DDR (Tesmer et al., 2023). As cells divide repeatedly and telomeres continue to shorten, shelterin binding and telomere-loop (t-loop) formation are impaired, and this weakened protection leads to telomere dysfunction, cellular senescence or apoptosis (Shi et al., 2023). TRF2 overexpression has been reported to be present in a variety of malignant cancers, and its downregulation leads to cell death. Yin-da Qiu et al. showed that FKB04, a flavokawain B derivative, effectively inhibited TRF2 expression in hepatocellular carcinoma cells and also induced telomere shortening, increased the number of telomere-free ends, and led to the disruption of the T-loop structure (Qiu et al., 2024). These results suggest that TRF2 is a potential therapeutic target for hepatocellular carcinoma and indicate that FKB04 may be a selective small-molecule inhibitor of TRF2, which is expected to be used in the treatment of hepatocellular carcinoma. Mutations in TRF2 lead to changes in telomeric DNA topology, which initiates ATM-dependent DDR (Benarroch-Popivker et al., 2016). TRF1 and TRF2 form a homodimer that binds to double-stranded telomere DNA. TRF1 inhibits ATR signalling during S phase and otherwise induces a fragile telomere phenotype. TRF1 small molecule inhibitors (ETP-47228 and ETP-47037) inhibit TRF1 binding to DNA, induce DNA damage and inhibit lung cancer and glioblastoma progression, suggesting that TRF1 is a potential therapeutic target and that ETP-47228 and ETP-47037 small molecule inhibitors may be useful in treating lung cancer and glioblastoma (García-Beccaria et al., 2015; Bejarano et al., 2017). TRF2 also binds to RAP1 and inhibits the localisation of SLX4 and PARP1 to telomeres, thereby inhibiting NHEJ (Rai et al., 2016). It has been reported in the literature that Triazole-stapled peptides can block protein interactions between RAP1 and TRF2, thereby inhibiting HR (Ran et al., 2016). POT1 has been reported to co-localize with the ubiquitin-specific processing protease 7 (USP7) deubiquitinating enzyme within APB, which is capable of deubiquitinating and stabilising the POT1-targeted ubiquitin ligase, whereas testis-specific Y-coding-like protein 5 (TSPYL5) has recently been identified as a PML component and functions as a USP7 inhibitor (Episkopou et al., 2019). This suggests that TSPYL5 may be a therapeutic target for ALT-positive cancer types. The above suggests that targeting a reduction in DDR-related proteins on telomeres would reduce cancer progression. Although no reliable inhibitors are available for the above, their importance for the types of cancers in which ALT is used suggests that they should be considered in the development of future ALT-targeted therapies.