Harnessing DNA Double-Strand Break Repair for Cancer Treatment

Abstract

DNA double-strand breaks (DSBs) are highly deleterious, with a single unrepaired DSB being sufficient to trigger cell death. Compared to healthy cells, cancer cells have a higher DSB burden due to oncogene-induced replication stress and acquired defects in DNA damage response (DDR) mechanisms. Consequently, hyperproliferating cancer cells rely on efficient DSB repair for their survival. Moreover, augmented DSB repair capacity is a major cause of radio- and chemoresistance and, ultimately, cancer recurrence. Although inherited DDR defects can predispose individuals to develop certain cancers, the very same vulnerability may be therapeutically exploited to preferentially kill tumor cells. A paradigm for DNA repair targeted therapy has emerged in cancers that exhibit mutations in BRCA1 or BRCA2 tumor suppressor genes, conferring a strong defect in homologous recombination, a major and error-free DSB repair pathway. Clinical validation of such approaches, commonly described as synthetic lethality (SL), has been provided by the regulatory approval of poly(ADP-ribose) polymerase 1 inhibitors (PARPi) as monotherapy for BRCA1/2-mutated breast and ovarian tumors. In this review, we will describe the different DSB repair mechanisms and discuss how their specific features could be exploited for cancer therapy. A major emphasis is put on advances in combinatorial treatment modalities and SL approaches arising from DSB repair pathway interdependencies.

Keywords: BRCA; DNA polymerase theta; DSB repair; PARP inhibition (PARPi); alternative end joining (a-EJ); cancer therapy; homologous recombination (HR); synthetic lethality.

Figure 1

DSB repair pathways in mammalian cells. Two-ended DSBs are preferably repaired by two major competing pathways: classical non-homologous end joining (c-NHEJ) and homologous recombination (HR). In addition, DSBs can be subjected to alternative end joining [a-EJ, also referred to as DNA polymerase theta-mediated end joining (TMEJ)] or single-strand annealing (SSA). BRCA1 and 53BP1 are placed at the center of DSB repair pathway choice. Whereas, chromatin recruitment of 53BP1 drives c-NHEJ, BRCA1 antagonizes 53BP1 to channel DSB repair into HR. (i) C-NHEJ begins with Ku70-Ku80 (Ku) binding to DSB ends, followed by the recruitment of DNA-dependent protein kinase catalytic subunit (DNA-PKcs), forming the DNA-PK holoenzyme implicated in DNA synapsis. If necessary, DNA-PK coordinates limited processing of incompatible or chemically modified DNA ends by nucleases (e.g., Artemis) and other enzymes. The DNA ligase IV (LigIV)-XRCC4-XLF-PAXX complex executes the final ligation step. DNA end resection interferes with the default engagement of c-NHEJ by removing Ku from DNA ends, which is a critical step for initiating HR (ii). First, the MRE11-RAD50-NBS1 (MRN) complex senses the DSB and with the help of BRCA1 and CtIP promotes limited resection of the 5′ strand. Next, more extensive 5′-3′ resection by exonuclease 1 (EXO1), or by the Bloom's syndrome (BLM) helicase together with the DNA2 nuclease, generates long 3′ ssDNA overhangs that become rapidly coated with the RPA heterotrimer. The BRCA1-PALB2-BRCA2 complex disassembles RAD51 heptamers and loads monomeric RAD51 onto ssDNA, promoting RAD51 filament assembly. Template-dependent strand extension is followed by “synthesis-dependent strand annealing” (SDSA), resulting in a non-crossover gene conversion. Alternatively, capture of the second ssDNA by the D-loop forms a double Holliday junction intermediate, which can be resolved either as a non-crossover or as a crossover. (iii) SSA requires at least 20–25 base pairs (bp) of DNA sequence homology, which are typically found between repetitive elements (indicated as green boxes) in the genome. Subsequently, RAD52 promotes annealing of complementary ssDNA and leftover non-homologous flaps of the 3′ overhangs are cleaved by XPF-ERCC1. The factors that promote gap filling and ligation during SSA remain largely elusive. (iv) In contrast to SSA, a-EJ (or TMEJ) utilizes short microhomologies (MHs) of 2–20 bp (indicated as red boxes) to join the two DNA strands. PARP1 has been implicated in promoting DNA end synapsis and recruiting the specialized DNA polymerase θ (Polθ) to DSBs. Polθ stabilizes MH-mediated joints between the two DNA ends serving as primers for fill-in synthesis. 3′ flaps extending from the joints are removed by XPF/ERCC1. Flap endonuclease 1 (FEN1) has recently been implicated in the removal of 5′ flaps generated by Polθ-mediated strand displacement, while the DNA Ligase III (LigIII)-XRCC1 complex is essential for the final ligation step. Inset, bottom left: DSB repair pathway-specific inhibitors. Inhibition of c-NHEJ has so far been mainly achieved by targeting DNA-PK using different small molecule inhibitors. Strategies to inhibit a-EJ and SSA focus on targeting their respective DNA annealing factors Polθ and RAD52, while the primary target to disrupt HR is RAD51 (see text for more details).

Figure 2

Synthetic lethal (SL) strategies to target HR-deficient (HRD) tumors. (A) PARPi (red dot) trap PARP1 proteins on endogenous DNA lesions, including single-strand breaks or gaps. If not removed timely, trapped PARP1 blocks the replication machinery, leading to one-ended DSBs that need to be repaired by HR. In HRD tumors such as BRCA1- or BRCA2-mutated cancers, DSBs persist and accumulate, ultimately causing cell death due to SL. (B) HRD tumors are addicted to alternative, mutagenic DSB repair process (a-EJ and SSA) to sustain proliferation. Targeting Polθ (or FEN1) or RAD52 by small molecule inhibitors (red dots) would eliminate a-EJ or SSA, respectively, resulting in SL. (C) Apurinic/apyrimidinic (AP) sites (black dot) are one of the most frequent spontaneous lesions in DNA. If not timely removed by APE2, they have the potential to block DNA replication. Consequently, APE2 inhibition (red dot) would lead to massive accumulation of AP sites associated with increased rates of fork collapse and DSB formation. In healthy cells, HR can deal with those DSBs, promoting cell survival. In contrast, treating HRD tumors with APE2i would cause DSB-induced cell death due to SL.