Targeting Replication Stress Response Pathways to Enhance Genotoxic Chemo- and Radiotherapy

Abstract

Proliferating cells regularly experience replication stress caused by spontaneous DNA damage that results from endogenous reactive oxygen species (ROS), DNA sequences that can assume secondary and tertiary structures, and collisions between opposing transcription and replication machineries. Cancer cells face additional replication stress, including oncogenic stress that results from the dysregulation of fork progression and origin firing, and from DNA damage induced by radiotherapy and most cancer chemotherapeutic agents. Cells respond to such stress by activating a complex network of sensor, signaling and effector pathways that protect genome integrity. These responses include slowing or stopping active replication forks, protecting stalled replication forks from collapse, preventing late origin replication firing, stimulating DNA repair pathways that promote the repair and restart of stalled or collapsed replication forks, and activating dormant origins to rescue adjacent stressed forks. Currently, most cancer patients are treated with genotoxic chemotherapeutics and/or ionizing radiation, and cancer cells can gain resistance to the resulting replication stress by activating pro-survival replication stress pathways. Thus, there has been substantial effort to develop small molecule inhibitors of key replication stress proteins to enhance tumor cell killing by these agents. Replication stress targets include ATR, the master kinase that regulates both normal replication and replication stress responses; the downstream signaling kinase Chk1; nucleases that process stressed replication forks (MUS81, EEPD1, Metnase); the homologous recombination catalyst RAD51; and other factors including ATM, DNA-PKcs, and PARP1. This review provides an overview of replication stress response pathways and discusses recent pre-clinical studies and clinical trials aimed at improving cancer therapy by targeting replication stress response factors.

Keywords: DNA damage response; DNA replication stress; genotoxic cancer therapy; oncogenic stress; targeted therapy.

Figure 1

DNA-PK, ATM and ATR roles DSB repair by cNHEJ, HR, and replication fork restart. (A) Two-ended (frank) DSBs are shunted to cNHEJ if resection is blocked by 53BP1 (and RIF1), or to HR when resection is promoted by MRN and signaling through ATM. Ku70/80 binds unresected ends and recruits DNA-PKcs (D-PK) which tethers ends, and promotes end alignment, template-dependent and -independent nucleotide addition by Pol λ and Pol μ, respectively, and ligation by Lig4-XRCC4 and XLF to yield indel repair products. ATM phosphorylates/activates Chk2 which promotes cell cycle arrest and repair. ATM stimulates DSB repair by HR by phosphorylating targets that promote limited end resection by CtIP and MRE11, and extensive resection by EXO1 and DNA2-BLM. The resulting 3′ ssDNA extensions are bound by RPA, which is replaced by the HR recombinase RAD51 that catalyzes invasion of homologous duplexes (typically sister chromatids). Eviction of RAD51 allows repair synthesis to extend the invading strand across the DSB. The extended end releases from the donor duplex (grey) and anneals to complementary ssDNA on the opposite side of the DSB. Gap filling and ligation complete accurate HR repair. (B) Replication forks blocked by single-strand lesions or secondary structures, or stalled by nucleotide depletion or DNA polymerase inhibitors, causes decoupling of polymerase from the MCM helicase, which spools out ssDNA that is bound by RPA. ATRIP mediates the interaction between ssDNA-RPA and ATR, which leads to Chk1 activation that phosphorylates targets that effect several DNA damage checkpoint responses. Blocked or stalled forks may be cleaved by nucleases MUS81 or EEPD1 to yield one-ended DSBs that are resected to promote HR-mediated fork restart and prevent genome rearrangements due to cNHEJ rejoining of one-ended DSBs to ends of other DSBs.

Figure 2

Fork restart mechanisms. (A) Blocked forks may regress to form 4-strand, Holliday junction-like structure and a one-ended DSB. DNA synthesis (dashed/red arrow) using newly replicated strand as template allows the blocked end to extend beyond the blocking lesion. The fork is restarted once RECQ1 drives fork reversal by branch migration. (B) The one-ended DSB at regressed forks may be protected by RAD51, BRCA2, and other factors. Fork restart is mediated by RAD51-ssDNA filament invasion of the unreplicated duplex DNA. (C) Stalled forks may be cleaved by MUS81-EME2 (or EEPD1, not shown), creating a one-ended DSB. Resection creates ssDNA that is first bound by RPA and then RAD51, and the RAD51-ssDNA filament catalyzes strand invasion to restart the fork, similar to panel (B).