Cell division cycle 6 homologue (CDC6) is an important regulator of DNA replication in eukaryotic cells (52, 53) involved in replication complex assembly during G1 phase. Replication fork stall accumulation caused by RS triggers G2/M checkpoint activation. CDC6 promotes the response of the G2/M checkpoint (54) and is positively correlated with tumor progression. Decreased CDC6 expression in tumor cells effectively inhibits tumor cell growth and promotes apoptosis by preventing G1/S and S/G2 transition (55). CDC6 overexpression has been observed in radiation-resistant cells, contributing to an increase in radiation resistance in cancer cells (56). CDC6 downregulation enhanced cisplatin-resistant bladder cancer cell sensitivity in a clinical trial, which is also related to DSB damage (57). Therefore, CDC6 inhibition in tumor cells might be an effective target for enhancing tumor radiosensitivity. Although CDC6 has druggable sites for a chemical molecular, it is an essential protein in most cell lines that makes it difficult for clinical transformation (58). Thus, further study on the regulatory mechanism of CDC6 in radiation resistance will help to develop clinical practical drugs in the future.

T-lymphoid-activated killer (T-LAK) cell-derived protein kinase (TOPK) is a mitogen-activated protein kinase kinase-like kinase that plays an important role in cell cycle regulation. TOPK overexpression is a pathophysiological feature in different tumors (59).

TOPK knockdown does not change the radiation response of normal tissues but significantly enhances cancer cell radiosensitivity, and TOPK disruption may lead to tumor-specific radiosensitivity (60). Thus, TOPK, as a cancer-specific biomarker and biochemical target, may enhance the efficacy of cancer treatment while causing minimal damage to normal tissues (59). TOPK was found to enhance tumor radiosensitivity by enhancing intratumor RS (61). Further experiments demonstrated that TOPK helps to restart the stopped replication fork. However, when TOPK was depleted, increased levels of stalled replication forks were observed, with or without external DNA damage (61). Therefore, TOPK suppression increases internal replication damage. Owing to the preservation of irradiation-induced damage and reduced tolerance to RS, TOPK sensitizes cancer cells to radiotherapy.

TOPK interacts with CHK1 and cell division cycle 25 homologue C (CDC25C) complex (key participants in the replication of the damage induced) (61). It facilitates mitotic progression at the G2/M checkpoint via cyclin-dependent kinase 1 (CDK1), and also occurs in response to replication stressors (such as irradiation) by influencing the action of key intermediates such as CHK1 (61). Therefore, the synergistic effect of TOPK inhibition and radiotherapy is likely to produce DSBs after replication. However, unlike CHK1, the toxicity of TOPK inhibitors is limited in normal tissues due to low expression. Therefore, TOPK appears to be a promising target for further research.

Cell division cycle 20 homologue (CDC20) has important functions in chromosome segregation and mitotic exit. It is the target of the spindle assembly checkpoint (SAC) and the key cofactor of the anaphase-promoting complex or cyclosome (APC/C) E3 ubiquitin ligase, thus regulating APC/C ubiquitin activity on specific substrates for their subsequent degradation by the proteasome (62). CDC20 is overexpressed in tumor cells and acts as a poor prognostic factor in multiple cancers (63, 64). It further increased after radiation and has been reported to increase radiation resistance via regulating B-cell lymphoma-2 (Bcl-2)/Bcl-2-associated X protein (Bax), forkhead box proteins O1 (FoxO1), or myeloid cell leukemia sequence 1 (Mcl-1)/p-CHK1 in different cancer types (65–67). Suppression of CDC20 expression reverses the radioresistance (65–67). There are multiple available inhibitors of CDC20, including tosyl-L-arginine methyl ester (TAME), Pro-TAME, and apcin. Their main effects involve disrupting the APC-CDC20 interaction (68, 69). Some of them showed great efficacy in suppress tumor proliferating and metastasis (70, 71). However, there has been no evidence on the effects of the CDC20 inhibitors on radiosensitivity. Therefore, CDC20 could be a potential target as a radiosensitizer, but more evidence in future studies is needed.

As the first anti-apoptotic protein in the Bcl-2 family, Mcl-1 is regulated by the cell cycle and reach peak expression levels in the S/G2 phase. It acts as a functional switch in selecting between HR and NHEJ pathways after DNA damage (72). It blocks radiation-induced apoptosis and inhibits clonogenic cell death (73). Targeting Mcl-1 by a small molecule enhances RS sensitivity to cancer therapy (72). BAY1143572 downregulated Mcl-1 by inhibiting binding of HIF-1α to the Mcl-1 promoter (74). UMI77 is a selective inhibitor of Mcl-1 that dissociates Mcl-1 from the pro-apoptotic protein Bak and produced significant radiosensitization in pancreas cancers (75).

Poly (ADP-ribose) polymerases (PARPs) are involved in DDR and recruit DNA repair proteins to damaged sites by catalyzing ADP-ribosylation, leading to the formation of poly (ADP-ribose) polymers (76). PARP1, the most abundant PARP, plays a similar role to PARP2 in the DDR process and is an important regulator of fork reversal (77). Inhibition of PARP directly increases the speed of fork elongation and does not cause fork stalling, which contrasts with the accepted model in which inhibitors of PARP induce fork stalling and collapse. Aberrant acceleration of fork progression by 40% above the normal velocity leads to DNA damage (78).

However, the effects of PARP inhibitor do not directly decrease the expression of PARP. Rather, the inhibitor forces PARP to become stuck on DNA, thus preventing replication restart and causing RS-induced DNA damage (79). It was also linked to decreased replication fork length with greater ssDNA gaps, which in turn cause more genomic instability at G2/M (80). With all the evidence of PARP inhibitors in RS-induced DNA damage, researchers have reported on various preclinical models of combination therapy with PARP inhibitors and ionizing radiation (IR) (81). Olaparib, a PARP inhibitor that has been widely used in cancer treatment, has been reported to have strong tumor-specific radiosensitization effects (82, 83).

The RPA complex is one of the first responders to coordinate DNA replication (18, 84). It consists of three subunits, RPA1 (RPA70), RPA2 (RPA32), and RPA3 (RPA14), which are essential to protect ssDNA at replication forks and recruits DNA polymerases α, δ, and ϵ for the initiation and elongation steps of DNA replication (84). It has been reported that RPA1 phosphorylation upon RS decreases the ubiquitination of chromatin-loaded RPA1, leading to an accumulation of RPA1 on stalled replication forks. This helps the DNA-binding domains of RPA2 to bind with RPA1-coated ssDNA, thus contributing to increased RPA2 binding stability (85). Loss of RPA accelerates fork breakage, whereas overexpression of RPA is sufficient to delay a “replication catastrophe” (86). It also plays an important role in DDR in relation to the HR pathway (87). Furthermore, overexpression of RPA significantly increases the radiation resistance in multiple cancer types (88–90). However, there has been no reported inhibitors of RPA because it is an essential protein to all cells. Furthermore, it is a downstream factor of ATR, and thus the regulation of ATR may produce similar effects (86). RING finger and WD repeat domain 3 (RFWD3)-mediated ubiquitination of RPA helps to remove RPA from the damage site, which is a crucial step for HR (91), and thus provides a possible target for increasing radiation sensitivity via ubiquitination regulation.

DNA topoisomerase II-binding protein 1 (TopBP1) serves as a scaffold to assemble protein complexes in a phosphorylation-dependent manner via its multiple breast cancer C-terminal (BRCT) repeats. It is repurposed to scaffold different processes dependent on cell cycle-regulated changes in phosphorylation of target proteins (92). It is known to form phase-separated nuclear condensates that amplifies ATR activity to CHK1 and slow down replication forks (93). TopBP1 also stabilized bloom syndrome helicase (BLM) to maintain genome stability (94). It is often overexpressed in cancer and can bypass control by CDK2 to interact with treslin, leading to enhanced DNA replication (95).

However, it has been reported that at low levels, TopBP1 activates ATR/CHK1, but once TopBP1 protein accumulates above an optimal level, it paradoxically leads to lower activation of ATR/CHK1. This is due to the perturbation of ATR-TopBP1 interaction and ATR chromatin loading by excessive TopBP1. Depletion of TopBP1 in some specific cancer cells enhanced ATR/CHK1 activation and S-phase checkpoint response after RS (96). Thus, simply inhibiting TopBP1 may lead to unexpected results, which makes it not an ideal target for radiation sensitization.

ATR of the phosphoinositide 3-kinase (PI3K) family is a central regulator of RS. After ssDNA fragments are coated with PRA, ATR and ATR-interacting protein are recruited and activated. It further phosphorylates various proteins, including CHK1 kinase, which inhibits mitotic entry and dormant origin activation. Mitotic entry is inhibited by CDC25 phosphatase phosphorylation, which prevents subsequent mitotic CDK activation (97). Cancer genome sequencing showed a very low ATR or CHK1 mutation or deletion frequency. Instead, these genes are often amplified in cancer cells, probably because they need to process high levels of RS to survive. ATR and CHK1 inhibition can increase RS, leading to mitotic catastrophes that trigger cell death (98). Furthermore, inhibition of ATR-related signaling pathways can increase cell apoptosis and effectively improve tumor radiosensitivity (99). ATR inhibitors, such as AZD6738 and VE-821 as well as the CHK1 inhibitor SAR-020106 were effective radiosensitizers in preclinical studies (100–102). An ongoing phase I clinical trial (NCT03188965) is assessing the safety profile of ATR inhibitors (BAY1895344) (103).

RAD51 is a master regulator of DNA replication and plays important roles in DSB repair, RS, and mitosis (104). RAD51 is a core factor in overcoming RS by slowing or stalling replication forks, which threatens replication integrity (105). It facilitates fork inversion, protects reverse forks, repairs and restarts broken replication forks, and post-replication gap filling (104, 106).

RAD51 inhibition may lead to increased tumor radiosensitivity, and it has been reported as a potential target of berberine in osteosarcoma radiosensitization (107). Valproate was found to increase tumor tissue cell radiosensitivity by increasing levels of RFWD3 and inhibiting RAD51 (108). The inhibition of nucleophosmin1 (NPM1) by YTR107, a small molecule that binds with NPM1, inhibits pentamer formation and represses RAD51 formation after IR. The synergistic effect of YTR107 and the PARP1/2 inhibitor ABT-888 increased RS and radiation-induced cell mortality (109).

BLM is a 3’-5’ ATP-dependent RecQ DNA helicase that is one of the most essential genome stabilizers involved in the regulation of DNA replication, recombination, and both homologous and non-homologous pathways of DSB repair (110). It interacts with topoisomerase IIIα (TOP3A), RecQ-mediated genome instability (RMI) 1, and RMI2 to form the BLM-Topoisomerase IIIα-RMI1-RMI2 (BTR) complex, which dissolves double Holliday junctions to produce non-crossover HR products. It also promotes DNA-end resection, restart of stalled replication forks, and processing of ultra-fine DNA bridges in mitosis (111). BLM helicase-deficient cells exhibit multiple defects in DNA replication, including accumulation of abnormal DNA replication intermediates, slower replication fork velocity, and excessive firing of dormant origins, thus exhibit increased levels of chromatid breakage and HR (112). It interacts directly with both RAD51 and RPA, and the function in DNA replication is regulated by sumoylation (113).

The high expression of BLM is a poor prognostic biomarker for multiple cancers (114, 115). Biallelic pathogenic variants in BLM cause bloom syndrome with severe pre- and postnatal growth deficiency, immune abnormalities, sensitivity to sunlight, insulin resistance, and a high risk for many cancers that occur at an early age (116). The symptoms of bloom syndrome including sensitivity to ultraviolet damage, which is similar to radiation, provide the possibility of transforming this genomic defect into a treatment sensitizer. ML216 is a small molecule inhibitor of BLM, and inhibits cell proliferation of BLM-proficient cells and increases the frequency of sister chromatid exchanges (117). Though there has been no data published on the links between a BLM inhibitor and radiation sensitivity till now, it is a promising target worth further research.

When ssDNA accumulation at stalled replication forks activates ATR, it phosphorylates CHK1, which in turn activates WEE1 kinase and inhibits CDC25 phosphatase. While WEE1 inhibits CDKs, the key drivers of cell cycle progression, by phosphorylating the conserved threonine 14 (Thr14) and tyrosine 15 (Tyr15) residues, CDC25 activates CDKs by dephosphorylating the same residues (118). Elevated WEE1 expression reduces RS and activates G2/M checkpoints, conferring cell resistance to CHK1 inhibitors (98). Recently, it has been reported that the ATR-WEE1 module inhibits the MOS4-associated complex (MAC) to regulate RS responses (118).

The evidence suggests that WEE1 inhibition impairs the RS response activated by ATR, and thus increases tumor cell radiosensitivity (119). WEE1 kinase inhibitors sensitize tumor cells to proton and X-ray irradiation by inducing RS, independent of TP53 mutation status, such as AZD1775 (120–122). Clinical trials have shown that the WEE1 inhibitor adavosertib could potentiate the efficacy of RT; however, its clinical application is limited by its unfavorable safety profile (123).

The p53 signaling pathway plays a key role in determining radiosensitivity in normal tissues but is often inactivated during cancer. Loss of p53 in tumor cells allows them to escape cell cycle arrest and apoptosis checkpoints and promotes the growth of early-stage cancer cells by skipping the cell cycle checkpoint caused by RS (125). During DNA replication, IR-induced DNA damage stalls replication forks, and single-strand breaks (SSBs) can be transformed into DSBs, thereby activating the ataxia telangiectasia mutated (ATM)/ATR pathway. ATM and ATR phosphorylate p53 to increase its stability and activate target genes. RS induced by chemotherapy drugs such as trifluridine leads to cell senescence or apoptosis of tumor cells according to the state of p53 (126). Acetylation of p53 may modulate cancer cell radiosensitivity, which provides a promising strategy for radiosensitization (127).

Meiotic recombination 11 (MRE11), the core of the MRE11/RAD50/NBS1 (MRN) complex, is involved in DNA break end detection, phosphorylation-dependent signal amplification, and DSB repair (128). The complex is critical for ATM activation of DSBs and downstream activation of G2/M and p53-dependent G1/S cell cycle checkpoints (129, 130). MRE11 also has endonuclease and exonuclease activities residing in the phosphodiesterase domain. These nuclease activities are crucial for the pathway choice of HR and NHEJ (131). Cancer cells rely on DNA repair for survival during cancer therapies, and thus MRE11 might be a promising synergistic therapeutic target.

Dysfunction of it in neoplastic breast tumors results in the accumulation of R-loops, replication-associated DSB, abundance of genomic deletions, and uncontrolled proliferation (132). Evidence suggests that its expression in cancer cells is critical for radioresistance (133). Low MRE11 expression in colorectal cancer cells reduced phosphorylated DNA-PKcs expression and further increases tumor radiosensitivity (134). There are different small and large molecular inhibitors targeting MRE11. Mirin is the first inhibitor found to specifically target MRE11 exonuclease activity with radiosensitizing properties (135). Lung cancer cells treated with Selenium, which is an essential trace element, showed decreased expression of MRE11 and significantly reduced colony formation relative to IR (136). OBP-301, with the insertion of the human telomerase reverse transcriptase (hTERT) promotor, also showed reduced MRE11 expression and thus enhanced radiosensitivity of lung cancer cells (137). Therefore, MRE11 inhibitors are clinically significant for enhancing radiosensitivity, and several clinical trials investigating their potential are ongoing (131).

ATM kinase is a member of the PI3K-like protein kinase (PIKK) family with extensive roles in DDR signaling (138). Upon recruitment by the MRN complex to DSBs, ATM autophosphorylates at different serine sites resulting in the activation of CHK2, p53, and H2AX, which are involved in DNA repair processes and cell cycle arrest (139). The most important transducer of ATM signaling is CHK2, a kinase that signals to DNA repair, cell cycle arrest, and apoptosis. ATM phosphorylates CHK2 on threonine 68 (Thr68), thereby causing CHK2 dimerization and autophosphorylation of the kinase domain and is required for full activation (140).

ATM orchestrates the cellular DDR to cytotoxic DNA DSBs induced by radiation (141, 142). Overexpression of ATM indicates radiation resistance in breast cancer cells (143), whereas deficiency of ATM showed radiation sensitizer effects in multiple cancer types (144–147). Interestingly, more studies have focused on the radiation sensitizer effects that are dependent on the cell cycle and proliferation status (148, 149). After inhibition of proliferation, ATM status did not alter cell death or micronucleus formation after radiation, which suggests that ATM in endothelial cells was immaterial if a cell cycle block was present at the time of irradiation. It is consistent with other data showing that the effect of ATM on radiation sensitivity is more dependent on cell cycle regulation rather than the DDR pathway (148,149). Considering that ATM is a large protein with extensive regions of unknown function, the inhibition of its kinase activity may produce better synergistic effect on treatment. AZD0156, as a potent and selective bioavailable inhibitor of ATM, showed strong radiosensitizer effects in vitro and in a lung xenograft model (150). Specially, the ATM inhibitor AZD1390 is optimized for penetration of the blood-brain barrier with radiosensitizing effects on glioma and lung cancer cell lines, even in a brain metastasis model (141, 151). All of the evidence suggests that treatments targeting ATM may be promising in clinical trials.

Mouse double minute 2 (MDM2) protein is a major negative regulator of p53 (152). When activated, p53 suppresses tumors in response to cell damage by mediating cell proliferation, cell cycle arrest, DNA repair, metabolism, angiogenesis, senescence, and apoptosis (153). In normal cells, the self-regulating feedback loop between MDM2 and p53 controls p53 expression (154, 155). The rescue of p53 function in cancer cells by inhibiting the interaction between p53 and MDM2 restored cycle arrest and apoptosis (156). Furthermore, inhibition of MDM2 phosphorylation leads to cell apoptosis and cell cycle arrest, thus repressing tumor cell proliferation in esophageal cancer cells (157). Additionally, MDM2 inhibitors, such as MI-219, increase tumor cell radiosensitivity in a p53-dependent manner. MI-219 combined with radiation resulted in increased p53-dependent DNA damage (158). A novel small-molecule inhibitor, APG-115, was found to enhance gastric adenocarcinoma cell radiosensitivity by blocking the interaction between MDM2 and p53 (159). Therefore, blocking the MDM2/p53 pathway has broad application prospects for treating tumors and enhancing tumor radiosensitivity, especially for tumors with low TP53 mutation levels, such as those of myeloid leukemia.

DNA polymerase theta (POLQ) is a DNA polymerase that protects against error-prone transduction DNA synthesis and error-prone DSB (160). It is involved in a major DNA repair pathway that was initially named as alternative end-joining or microhomology-mediated end joining, and was later termed polymerase theta-mediated end joining because POLQ is indispensable in this process (161). POLQ overexpression reduces replication fork speed and impairs cell cycle progression (162). Furthermore, breast cancer related protein (BRCA) 2 and POLQ co-inhibition significantly improves tumor cell sensitivity to cisplatin (163). Reduced POLQ expression inhibits DSB repair and tumor cell survival. Several hepatocellular carcinoma cell lines (Huh7, HepG2, MHCC-92L, SK-HEP-1, and BEL-7404) with low POLQ expression after knockdown were found to be significantly sensitive to chemotherapeutic drugs (160). Depletion of POLQ in POLQ-dependent cancers (i.e., malignancies deficient in HR) leads to synthetic lethality. Furthermore, POLQ depletion was shown to synergize with PARP inhibition (164, 165), and the antibiotic novobiocin was recently reported as a selective POLQ inhibitor (166). Thus, combining novobiocin with radiotherapy should be a new research direction for targeting radioresistance.

BRCAs (including BRCA1 and BRCA2) are thought to be the predominant proteins involved in HR in the DDR pathway. In addition, as a master regulator of HR, BRCA1 and BRCA2 also mediate fork protection (167, 168). BRCA mutations increase the susceptibility to various cancer types, including breast, ovarian, prostate, and pancreatic cancers (167). It is also well known that mutations in BRCA result in synthetic lethality with PARP inhibition. The underlying mechanism includes HR deficiency and increasing replication gaps. PARP inhibition results in replication fork collapse, chromosomal instability, cell cycle arrest in G2, and subsequent apoptosis in BRCA-deficient cells (169). Therefore, targeting PARP has become a reliable therapeutic strategy for eliminating BRCA1/2-mutated malignancies at diverse sites including the breast, primary peritoneum, fallopian tubes, ovaries, and pancreas (also see section 4.1.2) (170).

It has been reported that BRCA-deficient tumors are more sensitive to chemotherapeutic agents that induce RS (171). Furthermore, mutations in BRCA1/2 enhance radiosensitivity, indicating the possibility of BRCA as a biomarker of radiation sensitivity (172, 173). Since BRCA1/2 are both large proteins and have complex multiple functions, the development of inhibitors directly targeting BRCA1/2 is difficult to achieve. Therefore, PARPi has been suggested to patients with BRCA1/2 mutations for the synergistic lethal effects. The function of PARPi in radiosensitization are summarized in 4.2.1. Further research may focus on inhibitors that specifically affect the function of BRCA.

The PI3K/protein kinase B (AKT)/mammalian target of rapamycin (mTOR) pathway activates the downstream mediator mTOR to translate specific mRNA transcripts (174, 175). They synergistically work with CHK1 to repress DSB-induced RAD51 foci, thus impairing the HR process and enhancing RS in tumor cells. In addition, PI3K/mTORi slows the fork speed by increasing cell division cycle 45 homologue (CDC45) to promote a new origin of replication, thus enhancing CHK1-induced RS (176). The PI3K/AKT/mTOR signaling pathway is hyperactivated or altered in many cancer types (177). Inhibition of the pathway reduces tumor cell radioresistance (178, 179). For example, dactolisib, a dual PI3K/mTOR inhibitor, causes cell cycle arrest in the G2/M phase and improves the radiosensitivity of DU145 cell lines. Dactolisib also inhibits radiation-induced DSB repair in glioblastoma (GBM) cell lines by inhibiting DNA-PKcs and ATM and improves the radiosensitivity of radioresistant prostate cancer cell lines (180).

Torin 2 is a special class of PI3K pathway drugs, which not only inhibits the cell cycle at G1/S but also interferes with S phase progression, causing ssDNA accumulation, DNA damage, and increased checkpoint signaling in triple-negative BRCA cells (181). Furthermore, the dual PI3K/mTOR inhibitor apitolisib (GDC-0980) was demonstrated to inhibit growth and induce apoptosis in human GBM cells (182).

Post-translational modification of the DNA replication machinery by ubiquitin and small ubiquitin-like modifier (SUMO) plays key roles in cell division, DNA replication/repair, signal transduction, and cellular metabolism (184). Recent research revealed that ubiquitin/SUMO pathways are essential regulators of DNA replication during initiation, the S phase or elongation, and DNA replication termination (185). SUMO/ubiquitin equilibrium at active DNA replication forks controls CDK1 activation. An increase in ubiquitination of the replisome results in premature disassembly of the replication machinery and generation of CDK1-dependent DNA damage in the S phase (186).

Our group has identified ubiquitination factors that affect radiation sensitivity. We showed that ubiquitin-specific protease 9X (USP9X) mediates lysine-specific demethylase 4C (KDM4C) deubiquitination, which activates transforming growth factor-β2 (TGF-β2)/Smad/ATM signaling to promote radioresistance in lung cancer (142). Furthermore, ubiquitin-conjugating enzyme E2O (UBE2O) facilitates tumorigenesis and radioresistance by promoting MAX interactor 1 (Mxi1) ubiquitination and degradation (187). The SUMO-specific protease (SENP) pathway is also involved in tumor radiation sensitization (188). SUMO E3 ligase PIAS4, which is an essential signal for p53-binding protein 1 (53BP1) loading to the damage site, promote radiation resistance by increasing DDR (189). Ring finger protein 4 (Rnf4), an E3 ubiquitin ligase that targets SUMO-modified proteins, target SUMOylated mediators of DNA damage checkpoint protein 1 (MDC1) and SUMOylated BRCA1 loading at sites of DNA damage. Rnf4-deficient cells and mice exhibit increased sensitivity to IR by suppressing DDR (190). These findings identify ubiquitylation/SUMO as possible radiosensitization targets, but further research is needed.

UPR is the master regulator of endoplasmic reticulum (ER) stress. A deficiency in UPR results in apoptosis (191). Recent research revealed the link between hypoxia-induced RS and UPR (192). The induction of RNA/DNA helicase senataxin (SETX) in hypoxia is reliant on the protein kinase R (PKR)-like ER kinase (PERK)/activating transcription factor 4 (ATF4) arm of the UPR (32). Hypoxia is present in the majority of human tumors and is associated with poor prognosis due to the protection it affords to radiotherapy and chemotherapy (27). As we described earlier (section 3.1), anti-hypoxia treatments provide additional radiation benefits through cell apoptosis, which establishes a link between UPR and radiation sensitivity.

UPR is widely involved in the establishment and progression of cancers, including BRCA, prostate cancer, and GBM multiforme (193). Elevated mitochondrial UPR markers (mtHSP70 and HSP60) are associated with poor prognosis in patients with lung adenocarcinoma, which is activated by Maf1 through ATF5. Suppressing IR-induced mitochondrial UPR activation by rapamycin resulted in increased sensitivity to IR-mediated cytotoxicity (194). ONC201, an UPR activator, reduced oxidative phosphorylation and thus impairs cell cycle arrest, and the inhibition of DNA repair factors after radiation also enhanced radiation-induced cell death (34). As a new concept of radiosensitization, the clinical significance of UPR still requires further studies.

Excessive RS or RS deficiency leads to the accumulation of replication blockage-derived DNA in the cytoplasm or the formation of micronuclei, resulting in activating the cyclic GMP-AMP (cGAMP) and the cGAMP receptor stimulator of the interferon gene (STING) pathway. cGAMP synthase (cGAS) is a DNA sensor that recognizes and binds with DNA fragments in the cytoplasm, enabling cGAMP synthesis. cGAMP subsequently activates STING. The activation of STING further increases interferon regulatory factor 3 (IRF3) and nuclear factor kappa-light-chain-enhancer of activated B cell (NF-κB) levels (198). IRF3 and NF-κB act as transcription factors to trigger the transcription of IFN-I and cytokines (199). Apart from cGAS, γ-interferon-inducible protein-16, a cytosolic DNA sensor, can detect both self and non-self dsDNA to promote IRF3 and NF-κB-dependent interferon production via STING (195, 200, 201). IFN-1 plays a crucial role in both basal and therapeutic-induced immune responses to cancer. It is a potent immune cell activator, resulting in the activation and maturation of antigen presenting cells (198). The promotion of dendritic cell migration to the tumor site and their maturation depends on IFN-1 signaling (202, 203). Innate immune cells respond to IFN-1 by increasing antigen presentation and the production of immune response mediators, such as cytokines and chemokines. These events help in antigen presentation and chemokine production in innate cells as well as induce antibody production and enhance T-cell responses (198).

Innate immunity activation by tumor cells is a complex phenomenon. As we mentioned above, cancer cells usually experience higher RS, leading to more cytoplasmic DNA and micronuclei formation. They activate innate immunity by secreting IFN-1 via the cGAS-STING pathway, exocrine exosomes, or extracellular vesicles (EVs), which can be captured by immune cells for inducing a further immune response.

Cancer cells exposed to RS-inducing agents or deficient in RS response show the increased production of IFN-1 and proinflammatory cytokines that can foster an innate immune response (204, 205). One study found that the inhibition of the ATM/CHK2 DNA damage checkpoint axis led to excessive RS and cytosolic DNA accumulation, which subsequently activated the DNA sensor STING-mediated innate immune response in ARID1A-deficient tumors (206). Cytosolic DNA can also be released in exosomes or EVs (207, 208). Exosomes/EVs containing DNA works as DAMPs to innate immune cells. Study found that EVs and exosome dsDNA promoted inflammation via activating the STING pathway in macrophages (209).

The activation of STING in dendritic cells is essential for radiation-induced antitumor immunity (210). In contrast, cGAS-STING activation in tumor cells impairs HR in DDR, which promotes tumorigenesis (211). Moreover, cGAS can act as a decelerator of DNA replication forks, suppressing replication-associated DNA damage (212). The complex network mechanism made it hard to simply target or enhance cGAS-STING to reverse cancer treatment resistance. In contrast, high RS or RS-response deficiency always leads to simultaneous cell damage and immune activation. Hence, it would be a better choice for cancer treatment sensitization.