DNA Damage Response and DNA Repair in Cancer
The DNA repair and DNA damage response (DDR) pathway is frequently disrupted in cancer and is one of the hallmarks of cancer. Germline and/or somatic mutations in critical DNA repair/DDR genes cause predisposition to cancer and higher mutational burden in cancers, respectively.
Synthetic lethality is a genomic concept where the simultaneous disruption of two genes, typically in the same pathway, results in cellular death. Importantly, synthetic lethality has been shown to occur between genes and drugs, an approach that has been successfully exploited in tumors harboring DNA repair/DDR defects. This is exemplified by the approval of poly(ADP-ribose) polymerase (PARP) inhibitors for treating ovarian cancers with mutations in BRCA1/2. In this case, one gene is inactivated by mutation and the other is inactivated by the drug.
The DNA Repair Process
Cells constantly deal with damage to their DNA that can originate from endogenous processes, such as DNA replication stress, or exogenous exposures such as ionizing radiation and chemotherapy drugs. DNA damaging agents give rise to different types of DNA damage, and failure to repair the damage can result in a number of possible devastating consequences to the cell, including genetic instability and accumulation of mutations promoting tumorigenesis. Cells have therefore developed intricate repair mechanisms to deal with the different types of possible DNA lesions that arise; all with the goal of protecting genomic stability.
The major types of DNA repair mechanisms are:
Direct reversal represents the simplest form of DNA repair since it depends mostly on the activity of a single protein without the need for nucleotide removal, resynthesis, or ligation. For instance, O6-methylguanine (O6-mG) is a harmful genetic lesion induced by alkylating mutagens. The presence of alky groups at the O6 position of guanine cause G:C to A:T transitions and a block in gene transcription or DNA replication. In the direct reversal process, the alkyl group is removed by the enzyme O6-methylguanine DNA methyltransferase (MGMT) and the proper nucleotide is restored.
Base Excision Repair (BER)
BER is responsible for repairing small but highly mutagenic DNA lesions that are a significant threat to genomic fidelity and stability. These DNA lesions can be caused by ionizing radiation as well as endogenous mutagens generated from metabolic events such as oxidation, methylation, deamination, or spontaneous loss of a DNA base pair. The BER pathway is initiated by one of eleven DNA glycosylases which removes the damaged base. Following base excision, a different set of proteins fill the exposed gap with a single base (short-patch pathway) or multiple bases (long-patch pathway).
Nucleotide Excision Repair (NER)
NER is perhaps the most flexible mechanism in terms of the diversity of lesions that are recognized and repaired. The most significant of these lesions are pyrimidine dimers induced by UV light (cyclobutane pyrimidine dimers and 6-4 photoproducts), and other NER substrates include bulky chemical adducts, DNA intra-strand crosslinks, and some forms of oxidative damage. These types of lesions cause both a helical distortion of the DNA duplex and a modification of the DNA chemistry, both of which are hallmark features recognized by NER.
NER is a complex multi-step process involving a large network of more than 30 proteins. Beginning with recognition of the damaged base, the DNA duplex is then unwound, and the damaged base(s) is removed by an excision repair complex which is then filled in and ligated.
DNA Mismatch Repair (MMR)
The MMR pathway plays an essential role in the correction of replication errors such as base-base mismatches and insertion/deletion loops (IDLs) that result from DNA polymerase misincorporation of nucleotides and template slippage, respectively. MMR also corrects mismatched pairs or “mispairs” generated by the spontaneous deamination of 5-methylcytosine and heteroduplexes formed following genetic recombination.
Defects in this pathway result in a "mutator" cellular phenotype characterized by elevated frequencies in spontaneous mutations and increased microsatellite instability (MSI). Mutations in several human MMR genes cause a predisposition to hereditary nonpolyposis colorectal carcinoma (HNPCC), as well as a variety of sporadic tumors that display MSI.
Double-Strand Break (DSB) Repair
DSBs are highly toxic genetic lesions that pose a serious threat to cellular homeostasis since they can affect transcription, replication, and chromosome segregation. DSBs are caused by a variety of exogenous factors, such as ionizing radiation and certain genotoxic chemicals, and endogenous factors, such as reactive oxygen species, replication of single-stranded DNA breaks, and mechanical stress on the chromosomes.
DSBs differ from most other types of DNA lesions mostly because they affect both strands of the DNA and therefore prevent use of the complementary strand as a template for repair (i.e. BER, NER, and MMR). Failure to repair DSBs can result in devastating chromosomal instabilities that can lead to dysregulated gene expression and increased risk of carcinogenesis.
Cells have evolved two distinct pathways of DSB repair: homologous recombination (HR), and non-homologous end joining (NHEJ). While a cell may opt to use either of these pathways to repair DSBs, the exact details of why a cell chooses one pathway over another remains unknown. Selection appears to be influenced by the cell cycle stage at the time the damage was acquired.
HR maintains genome stability by repairing DSBs, gaps, and restarting stalled replication forks. It is a relatively slow but error-free pathway that relies on the presence of a homologous sequence in the genome as a template to replace the damaged DNA segment.
Unlike HR, NHEJ does not require a DNA template (sister chromatid) for repair. Instead, NHEJ operates by modifying the free ends of DNA located on either side of the break by using various nucleases so that the ends become compatible (i.e. 3’-hydroxyl and a 5’-phosphate), followed by ligation with the enzyme DNA ligase 4. In contrast to HR, NHEJ is a relatively quick but intrinsically error-prone process, and its excessive use can lead to gene rearrangements, deletions, and mutations, all of which can cause post-replicative cells to be more vulnerable to DSBs.
DNA damage sensor proteins and DNA damage signaling proteins can both be targeted with a range of anticancer agents.
Drug Targeting of DNA Damage Sensor Proteins
DDR is essential for the activation of repair pathways and cell survival. DDR sensor proteins, which respond to a wide variety of DNA lesions, are key for initiating repair.
For DSBs, Ku and MRN represent the major sensor protein complexes. Ku is a protein heterodimer consisting of Ku70/Ku80, and it is part of the NHEJ machinery that immediately binds to DNA DSBs. Upon recognition and binding of DSBs, Ku recruits other proteins that assist in classical NHEJ repair.
The MRN complex is also important for initial detection of DSBs. After binding to the damaged site, MRN recruits the DNA-damage signaling kinase ATM where it becomes activated and triggers a cascade of signaling events that initiate DNA end-resection and promote repair by HR. A variety of other DNA-damage sensors have also been identified including the Fanconi anemia core complex, mismatch repair proteins, nucleotide excision repair proteins which are sensors of DNA inter-strand crosslinks, base-base mismatches or insertion-deletion loops and UV-induced photo-lesions.
PARP-1 is a key DNA damage sensor protein that has been successfully targeted with drugs largely due to the synthetic lethal relationship between PARP and BRCA. Synthetic lethality arises when simultaneous loss of function of two genes (often in the same pathway) results in cell death. The clinical success of targeting of PARP with small molecule inhibitors (e.g. olaparib, rucaparib, niraparib, and talazoparib) in BRCA-mutated ovarian cancer has provided proof-of-principle for exploiting synthetic lethality as a treatment strategy and has led to the development of other therapies targeting the DNA damage response.
Drug Targeting of DNA Damage Signaling Proteins
DDR signaling proteins trigger a wide variety of post-translational modifications and assembly of protein complexes that amplify and diversify the DNA damage signal so that the appropriate responses can be initiated, and can include: transcriptional changes, cell cycle checkpoint activation, alternative splicing, engagement of DNA repair processes, or in the context of massive damage, activation of cell senescence or apoptotic pathways.
The main proteins coordinating the signalling events of the DDR and novel drugs targeting this pathway are discussed below.
DNA protein kinase (DNA-PK) is required for proper DSB repair by NHEJ, which is the primary DNA repair pathway for DSBs in human cells that occur through all phases of the cell cycle. Small molecule inhibitors of DNA-PK kinase activity stabilize DNA-PK on DNA ends and subsequently impair NHEJ, likely also interfering with other repair processes, including HR by obstructing DNA end-resection. Loss of DNA-PK activity leads to reduced cell proliferation and initiation of caspase-mediated cell death.
One of the challenges associated with targeting DNA-PK has been selectivity due to structural similarities with other kinases. Given its role in NHEJ, drugs that target DNA-PK have been found to be more effective when combined with agents that induce replication-independent DSBs such as ionizing radiation and topoisomerase 2 inhibitors (e.g. doxorubicin, etoposide).
Several small molecules are currently in various stages of clinical development including MSC2490484A, VX-984, and CC-115.
ATM is a protein kinase that facilitates DSB repair and responds to DSBs throughout the cell cycle. ATM is predominantly activated through interactions with NBS1 of the MRN complex. It is the principal kinase responsible for the phosphorylation of histone H2AX, which occurs rapidly following DSBs and serves as a foundation for the assembly of the DNA repair machinery. ATM inhibition has been demonstrated to make cells very sensitive to ionizing radiation and DSB-inducing agents (e.g. etoposide, camptothecin and doxorubicin).
A phase I trial of the ATM inhibitor AZD0156 is currently being evaluated as monotherapy and in combination with the PARP inhibitor olaparib as well as other cytotoxic agents including irinotecan.
ATR is activated by replication protein A (RPA) bound at sites of ssDNA such as stalled replication forks or following 5’-3’degradation of one of the DNA strands (i.e. DNA-end resection) during the early stages of HR.
Berzosertib (also known as M6620 and VX-970) is a first-in-class ATR inhibitor, with preclinical data demonstrating chemosensitization of lung cancer cells predominantly to chemotherapeutics. This results in replication fork collapse, such as cisplatin and gemcitabine in vitro, and increased antitumor activity in combination with cisplatin in vivo.
CHK1 is a protein kinase that lies downstream of ATR and functions as a critical regulator of the intra-S and G2-M cell cycle checkpoints. Given the role of CHK1 in mediating cell cycle arrest following DNA damage, it is not surprising that CHK1 inhibitors appear to be most effective when combined with agents inducing DNA damage during DNA replication. Their clinical development has therefore focused on their use in combination with such drugs.
MK8776 is a potent and selective CHK1 inhibitor that has been demonstrated to be well tolerated as a monotherapy as well as in combination with gemcitabine. More recently, the CHK1 inhibitor prexasertib also demonstrated single agent and combination activity in preclinical and clinical trials.
The WEE1 protein kinase, which works in parallel with CHK1, plays a critical role in activating the G2-M checkpoint through the regulation of cyclin dependent kinases. However, unlike CHK1, WEE1 is not directly regulated by DNA damage but it is required for physiological cell cycle progression.
As for WEE1 inhibitors, it was believed that their mechanism-of-action led to mitotic catastrophe by preventing the activation of the G2-M checkpoint as a result of inappropriate CDK1/CCNB1 activation. However, more recent data have demonstrated that WEE1 inhibition also generates replication-dependent DNA damage in cells due to aberrant DNA replication through CDK2 inhibition.
The first-in-class WEE1 kinase inhibitor AZD1775 has been shown to increase the cytotoxic effects of a range of DNA damaging agents and has demonstrated single agent activity in preclinical models. A recent phase 1 study found AZD1775 in combination with a topoisomerase inhibitor was tolerable and the agent has transitioned to phase 2 studies.
While NHEJ and HR are the major DSB repair pathways, the importance of alternative homology-directed repair mechanisms is increasingly being recognized. For instance, HR-deficient cells are known to rely on error-prone microhomology-mediated end-joining (MMEJ) for DNA repair and survival. The DNA polymerase activity of POLQ (DNA polymerase theta) is required for gap-filling during MMEJ, and POLQ also prevents hyper-recombination by limiting RAD51 accumulation at resected DNA ends. Therefore, POLQ is an attractive drug target, especially in the context of HR-deficient tumors.
The development of small molecule inhibitors that disrupt protein-protein interactions of the RAD51 recombinase family are also under development and may be particularly effective against HR deficient tumors. A recent analysis identified fourteen compounds (other than PARP1/2 inhibitors) targeting DDR and in clinical development, with additional agents being evaluated in the preclinical setting.
DNA Damage Response (DDR) Combination Strategies
The ability to effectively respond to DNA damage is often lost in many types of cancers and targeting this pathway has been clinically validated in subsets of patients such as those with BRCA1/BRCA2 mutations treated with a PARP inhibitor. To enhance therapeutic efficacy, DDR inhibitors can be combined with drugs targeting other DDR proteins or completely different signaling pathways with the goal of blocking multiple pathways that cancer cells rely on for survival.
For example, clinical trials are currently underway testing PARP inhibitors in combination with small molecules targeting other members of the DDR pathway including AZD6738 (ATR), AZD0156 (ATM) or AZD1775 (WEE1) as well as ATR inhibition in ATM-deficient tumors, targeting WEE1 in cyclin E or MYC amplified tumors, and POLQ inhibitors in HRD- or NHEJ-deficient tumors. Preclinical studies have also shown targeting DDR proteins may be beneficial in tumors with deregulated oncogene expression as strong oncogenic signals can induce replicative stress.
DDR inhibitors can also be combined with standard of care drugs, such as the use of PARP inhibitors to enhance the effects of platinum-based agents and other studies evaluating other DDR inhibitors including CHK1/2 and WEE1 inhibitors in combination with chemoradiation.
Finally, there is significant scientific rationale and clinical evidence that DDR and immune responses are connected and potentially synergistic. As our understanding of the interactions between DNA damage, DDR, and the immune response increases, there may be potential for increasing clinical efficacy of immunotherapies by combining them with DDR inhibitors and/or radiation as sensitizers.
For example, when genetic lesions are not repaired this can lead to dramatic alterations in mutational load and neoantigen expression on the surface of tumor cells. Preclinical studies suggest combining DNA repair targeted therapies with immune checkpoint inhibitors can have synergistic effects such as dual CTLA-4 and PARP blockade which led to reduced tumor burden and increased survival in a mouse model of BRCA-mutant ovarian cancer.
In the clinical setting, early studies have supported the safety of combining PARP inhibitors with anti-PD/PD-L1 agents20 and multiple trials are underway exploring combinations with other DDR inhibitors including those targeting ATR.
While cancer cells may benefit from defects in the DDR pathway, the presence of other functioning DNA repair systems can enhance survival. Drugs that target the DDR have been clinically validated in subsets of patients and different combination strategies are actively under study to inhibit multiple pathways that cancer cells rely on for survival. By impeding DNA repair, DDR inhibitors are ideally suited to combination therapies that can improve the efficacy of radiation, chemotherapy, and immunotherapy.