DNA damage response (DDR)
Objective to explore a new anti-cancer therapeutic strategy targeting various cellular DNA damage response (DDR) and DNA repair pathway

DNA damage response and DNA repair in cancer

When cancer occurs, DNA repair and DNA damage response (DDR) pathways are often destroyed, which is one of the signs of cancer. Germline and / or somatic mutations in key DNA repair / DDR genes lead to susceptibility to cancer and high mutation burden in cancer, respectively.

Synthetic lethality is a genomic concept, which usually destroys two genes in the same pathway, leading to cell death. Importantly, synthetic lethality has been shown to occur between genes and drugs, and this method has been successfully applied to tumors carrying DNA repair / DDR defects. The approval of poly (ADP ribose) polymerase (PARP) inhibitors for the treatment of ovarian cancer with BRCA1 / 2 mutation is an example. In this case, one gene is inactivated by a mutation and the other by a drug.

DNA repair process

Cells constantly respond to DNA damage caused by endogenous processes (such as DNA replication pressure) or exogenous exposure (such as ionizing radiation and chemotherapy drugs). DNA damaging agents can lead to different types of DNA damage, and failure to repair the damage will cause a series of potentially devastating consequences to cells, including gene instability and mutation accumulation that promotes tumor formation. As a result, cells have developed complex repair mechanisms to deal with different types of DNA damage that may occur; all of these are to protect the stability of the genome.

The main types of DNA repair mechanisms include:

Direct repair

Direct repair is the simplest form of DNA repair because it mainly depends on the activity of a single protein and does not require nucleotide removal, re synthesis or ligation. For example, O6 methylguanine (O6 mg) is a harmful gene damage caused by alkylation mutagens. The presence of alkyl groups at guanine O6 site leads to G: C to a: T transition and gene transcription or DNA replication block. In the direct repair process, the alkyl group was removed by O6 methylguanine DNA methyltransferase (Mgmt) and the appropriate nucleotides were recovered.

Base excision repair (BER)

BER is responsible for repairing small but highly mutagenic DNA damage that poses a significant threat to genomic fidelity and stability. These DNA damage can be caused by ionizing radiation, metabolic activities such as oxidation, methylation, deamination, or spontaneous loss of DNA base pairs. BER is activated by one of 11 DNA glycosidases that take out the damaged base. After base excision, a group of different proteins fills the exposed gap through a single base (short patch pathway) or multiple bases (long patch pathway).

DNA mismatch repair (MMR)

The MMR pathway plays an important role in correcting replication errors, such as base base mismatch and insertion / deletion loop (IDL) caused by DNA polymerase mismatch and template slip, respectively. MMR also corrected the mismatch or "mismatch" caused by the spontaneous deamination of 5-methylcytosine and heteroduplex nucleic acid molecules formed after gene recombination.

Defects in this pathway result in a "mutant" cell phenotype characterized by an increase in the frequency of spontaneous mutations and an increase in microsatellite instability (MSI). Mutations in several human MMR genes lead to susceptibility to hereditary nonpolyposis colorectal cancer (HNPCC) and a variety of sporadic tumors with MSI.

Repair of DSB

DSB is a kind of highly toxic gene damage, which poses a serious threat to the automatic regulation of cells, because they can affect transcription, replication and chromosome separation. DSB is caused by a variety of exogenous factors, such as ionizing radiation and some genotoxic chemicals, as well as endogenous factors such as reactive oxygen species, replication of single strand DNA breaks and mechanical stress on chromosomes.

DSB is different from most other types of DNA damage, mainly because they affect the double strand of DNA, thus preventing the use of complementary chains as templates for repair (i.e., BER, NER, and MMR). Failure to repair DSB can lead to devastating chromosomal instability, resulting in abnormal gene expression and increased risk of cancer.

Cells have evolved two different DSB repair pathways: homologous recombination (HR) and non homologous end joining (NHEJ). Although cells may choose to use either of these pathways to repair DSB, the reason why cells choose one pathway rather than the other is still unknown. At the time of injury, selection seems to be influenced by cell cycle stages.


HR maintains genomic stability by repairing DSBs, gaps and restarting stalled replication forks. This is a relatively slow but error free pathway, which relies on homologous sequences in the genome as templates to replace damaged DNA fragments.


Unlike HR, NHEJ does not require DNA templates (sister chromatids) for repair. In contrast, NHEJ becomes compatible (i.e., 3 ′ - hydroxy and 5 ′ - phosphate) by modifying the free end of DNA on either side of the cleavage by using different nucleases, and then connects with DNA ligase 4. Compared with HR, NHEJ is a relatively fast but essentially error prone process. Overuse can lead to gene rearrangement, deletion and mutation, all of which can lead to more vulnerable cells to DSB after replication.

DNA damage receptor protein and DNA damage signal protein can be used as targets of a series of anticancer drugs.

Drug targeted therapy of DNA damage receptor protein

DDR plays an important role in the activation of repair pathway and cell survival, and DDR receptor proteins that respond to various DNA damage are the key to initiate repair.

For DSB, Ku and MRN are the main receptor protein complexes. Ku is a protein heterodimer composed of Ku70 / Ku80, which is part of NHEJ mechanism and can immediately bind to DNA DSB. After recognizing and binding to DSB, Ku absorbs other proteins to assist in classical NHEJ repair.

MRN complex is also important for the initial detection of DSB. After binding to the damaged site, MRN absorbs DNA damage signal kinase ATM, activates and triggers a series of signal events, initiates DNA terminal excision and promotes HR repair. A variety of other DNA damage receptors have been identified, including Fanconi anemia core complex, mismatch repair protein and nucleotide excision repair protein, which are receptors for DNA cross-linking, base base base mismatch or insertion deletion loops, and UV induced light damage.

PARP-1 is a key DNA damage receptor protein. Due to the synthetic lethal relationship between PARP and BRCA, PARP has been used as a drug target to a large extent. When the loss of function of two genes (usually in the same pathway) leads to cell death, synthetic death occurs. Clinical PARP targeting therapy has been successfully achieved in BRCA mutant ovarian cancer by small molecule inhibitors (such as olapani, nilapali, nilapali and terazopani), which provides the principle verification of synthetic lethality as a treatment strategy, and other therapies targeting DNA damage response have been developed.

Drug targeted therapy of DNA damage signaling proteins

DDR signaling proteins trigger various post-translational modifications and protein complex assembly that amplify and diversify DNA damage signals, so as to initiate appropriate responses, and can include: transcriptional changes, cell cycle checkpoint activation, alternative splicing, participation in DNA repair process, or activation of cell aging and apoptosis pathways in the context of large-scale damage.

The main proteins that coordinate DDR signaling events with new drugs targeting this pathway are discussed below.

NHEJ needs DNA protein kinase (DNA PK) to repair DSB normally, which is the main DNA repair pathway of DSB in human cells at all stages of cell cycle. Small molecule inhibitors of DNA-PK kinase activity stabilize DNA-PK at the end of DNA, then damage NHEJ, and may interfere with other repair processes, including HR achieved by blocking DNA terminal excision. Loss of DNA-PK activity leads to decreased cell proliferation and initiation of caspase mediated cell death.

Because of its structural similarity to other kinases, one of the challenges associated with targeting DNA-PK is selectivity. In view of its role in NHEJ, it has been found that DNA-PK targeted drugs are more effective when used in combination with drugs that induce replication independent DSB, such as ionizing radiation and topoisomerase 2 inhibitors (such as doxorubicin and etoposide).

Some small molecules are currently in different stages of clinical development, including msc2490484a, vx-984 and CC-115.


ATM is a DSB protein kinase that promotes DSB repair and response throughout the cell cycle. ATM is activated mainly by NBS1 interaction with MRN complex. It is the main kinase responsible for histone H2AX phosphorylation, which occurs rapidly after DSB and serves as the basis for assembling DNA repair mechanisms. ATM inhibition has been shown to make cells very sensitive to ionizing radiation and DSB drugs such as etoposide, camptothecin and doxorubicin.

Currently, phase I trials of ATM inhibitor azd0156 are being evaluated as monotherapy and in combination with PARP inhibitor olapani and other cytotoxic drugs, including irinotecan.


At the early stage of HR, ATR is activated by replication protein A (RPA) bound to the ssDNA site, such as a stagnant replication fork, or subsequent 5 '- 3' degradation in one of the DNA strands (i.e., DNA terminal excision).

Berzosertib (also known as m6620 and vx-970) is the first ATR inhibitor. Preclinical data show that lung cancer cells are mainly sensitive to chemotherapy drugs. This leads to the collapse of replication forks, such as cisplatin and gemcitabine (in vitro), and increases antitumor activity when combined with cisplatin (in vivo).


CHK1, a protein kinase downstream of ATR, is a key regulator of S and G2-M cell cycle checkpoints. Given the role of CHK1 in mediating cell cycle arrest after DNA damage, it is not surprising that CHK1 inhibitors appear to be most effective when used in combination with drugs that induce DNA damage during DNA replication. Therefore, their clinical development focuses on the combined use of these drugs.

Mk8776 is an effective selective CHK1 inhibitor. It has been proved that both monotherapy and gemcitabine are well tolerated. Recently, in preclinical and clinical trials, the CHK1 inhibitor, prexasertib, has also shown single and combined activity.


The Wee1 protein kinase, which acts in parallel with CHK1, plays an important role in activating G2-M checkpoint by regulating cyclin dependent kinase. However, unlike CHK1, Wee1 is not directly regulated by DNA damage, but it is a protein kinase necessary for physiological cell cycle process.

For Wee1 inhibitors, we believe that their mechanism of action is to prevent the activation of G2-M checkpoint as a result of inappropriate CDK1 / ccnb1 activation, leading to mitotic catastrophes. However, recent data indicate that CDK2 inhibition leads to abnormal DNA replication, and Wee1 inhibition also leads to replication dependent DNA damage in cells.

It has been proved that the first Wee1 kinase inhibitor azd1775 can increase the cytotoxicity of a series of DNA damaging agents, and the single drug activity has been proved in preclinical model. Recently, a phase I study found that the combination of azd1775 and topoisomerase inhibitors was tolerable, and has been transferred to phase II study.

Although NHEJ and HR are the main DSB repair pathways, the importance of selective homologous recombination repair mechanism is increasingly recognized. For example, HR is known to lack the ability to rely on error prone microhomology mediated end joining (mmej) for DNA repair and survival. In mmej, the DNA polymerase activity of polq (DNA polymerase θ) is required for gap filling, and polq also prevents excessive recombination by limiting the accumulation of Rad51 at the ends of excised DNA. Therefore, polq is an attractive drug target, especially in HR deficient tumors.

Small molecule inhibitors that disrupt the protein-protein interaction of Rad51 recombinase family are being developed and may be particularly effective in HR deficient tumors. In a recent analysis, 14 compounds (other than PARP1 / 2 inhibitors) in clinical development and targeting DDR were identified, and other drugs were evaluated in the preclinical environment.

Joint strategy of DNA damage response (DDR)

In many types of cancer, the ability to effectively respond to DNA damage is often lost. Drugs targeting this pathway have been clinically proven in patient subsets, such as the use of PARP inhibitors in the treatment of BRCA1 / BRCA2 mutations. In order to improve the therapeutic effect, DDR inhibitors can be combined with drugs targeting other DDR proteins or completely different signaling pathways, in order to block the multiple pathways that cancer cells rely on for survival.

For example, clinical trials are currently examining the use of PARP inhibitors in combination with small molecules targeting other members of the DRR pathway, including azd6738 (ATR), azd0156 (ATM), or azd1775 (Wee1), as well as ATM deficiency of ATR inhibition in tumors, cyclin E or myc expansion, and the absence of polq inhibitors in tumors by HRD or NHEJ. Preclinical studies have also shown that targeting DDR protein may be beneficial for the treatment of tumors with aberrant gene expression, because strong carcinogenic signals can induce replication pressure.

DDR inhibitors can also be used in combination with standard therapeutic drugs, such as the use of PARP inhibitors to enhance the efficacy of platinum drugs, and other studies to evaluate the use of other DDR inhibitors (including CHK1 / 2 and Wee1 inhibitors) in combination with chemoradiotherapy.

Finally, there are important scientific evidence and clinical evidence that DDR and immune response are interrelated and have potential synergy. With the deepening of our understanding of the interaction between DNA damage, DDR and immune response, its combination with DDR inhibitors and / or radiation (as sensitizers) may improve the clinical efficacy of immunotherapy.

For example, when the gene damage is not repaired, the mutation load and the expression of new antigen on tumor cell surface will be greatly changed. Preclinical studies have shown that in mouse models with BRCA mutant ovarian cancer, the combination of DNA repair targeting therapy and immune checkpoint inhibitors can produce synergistic effects, such as dual CTLA-4 and PARP blocking, so as to reduce tumor load and improve survival rate.

In a clinical setting, early studies supported the safety of a combination of PARP inhibitors and anti Pd / PD-L1 drugs. At present, a number of experiments are being carried out to explore the combined use of other DDR inhibitors, including DDR inhibitors targeting ATR.


Although cancer cells may benefit from defects in the DDR pathway, there are other DNA repair systems that work to improve survival. Drugs targeting DDR have been clinically proven in patient subsets, and different combination strategies are being actively studied to inhibit multiple pathways that cancer cell survival depends on. By blocking DNA repair, DDR inhibitors are ideal drugs for combination therapy, which can improve the efficacy of radiation, chemotherapy and immunotherapy.

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