Targeting DNA repair mechanisms in cancer

Abstract

Preservation of genomic integrity is an essential process for cell homeostasis. DNA-damage response (DDR) promotes faithful transmission of genomes in dividing cells by reversing the extrinsic and intrinsic DNA damage, and is required for cell survival during replication. Radiation and genotoxic drugs have been widely used in the clinic for years to treat cancer but DNA repair mechanisms are often associated with chemo- and radio-resistance. To increase the efficacy of these treatments, inhibitors of the major components of the DDR such as ATM (ataxia telangiectasia mutated), ATR (ATM and Rad3-related), DNA-PK (DNA-dependent protein kinase, catalytic subunit), Chk1 (checkpoint protein 1) and Chk2 (checkpoint protein 2) have been used to confer radio- and/or chemosensitivity upon cancer cells. The elucidation of the molecular mechanisms of DNA repair and the discovery that tumors are frequently repair-deficient provide a therapeutic opportunity to selectively target this deficiency. Genetic mutations in the DNA repair genes constitute not only the initiating event of the cancer cell but also its weakness since the mutated gene is often needed by the cancer cell to maintain its own survival. This weakness has been exploited to specifically kill the tumor cells while sparing the normal ones, a concept known as ‘synthetic lethality’. Recent efforts in the design of cancer therapies are directed towards exploiting synthetic lethal interactions with cancer-associated mutations in the DDR. In this review, we will discuss the latest concepts in targeting DNA repair mechanisms in cancer and the novel and promising compounds currently in clinical trials.

Introduction

Genomic integrity is critical to organismal survival and is controlled by the DDR network, an elaborate signal transduction system that senses DNA damage and recruits appropriate repair factors (Harper & Elledge, 2007). This global signaling network senses different types of damage and coordinates a response that includes activation of transcription, cell cycle control, apoptosis, senescence, and DNA repair processes (Zhou & Elledge, 2000).

At the core of the DNA damage signaling apparatus are a pair of related protein kinases, ATM and ATR which are activated by DNA damage. ATM with its regulator the MRN (MRE11, Rad50-NBS1) complex senses double-strand breaks (DSBs) (Kastan & Bartek, 2004). ATR with its regulator ATRIP (ATR-interacting protein) senses single-strand DNA (ssDNA) generated by processing of DSBs, as well as ssDNA present at stalled replication forks. Both kinases then phosphorylate a plethora of downstream proteins to initiate a signaling cascade that includes many common substrates including Chk1 and Chk2 that start a secondary wave of phosphorylation events to amplify the signal (Fig. 1).

A recent large scale proteomic analysis of proteins phosphorylated in response to DNA damage on consensus sites recognized by ATM and ATR identified more than 900 regulated phosphorylation sites encompassing over 700 proteins (Matsuoka et al., 2007). This study identified a large number of functional modules, including developmental processes, immunity and defense, and intracellular protein traffic, that were far beyond the expected response to DNA damage and replication stress. This illustrated the extraordinarily broad landscape of the DDR and suggested that the DDR profoundly alters cellular physiology (Matsuoka et al., 2007).

Following DNA damage, DNA repair and cell cycle checkpoints are the main mechanisms of maintenance of genomic stability (Krempler et al., 2007). Several checkpoints are activated at different stages of the cell cycle. The G1/S and intra-S checkpoints prevent inappropriate DNA replication, whereas the G2/M checkpoint prevents cells with DNA damage from entering mitosis. As mentioned earlier, once activated, ATM and ATR phosphorylate a host of substrates, initiating a cascade that results in cell cycle arrest and DNA repair. Critical to the recruitment of DNA repair proteins to sites of DNA damage (nuclear foci), is phosphorylation of histone H2AX on Ser139 by ATM and ATR (Rogakou, 1998, Helt et al., 2005) leading to the accumulation of repair proteins in the DSB sites (Kouzarides, 2007, Taverna et al., 2007). Several proteins involved in DDR contain specific H2AX recognition domains such as forkhead-associated (FHA) domain (Durocher et al., 1999) and BRCT domains (BRCA1 C-terminal domain) (Manke et al., 2003, Falck et al., 2005) such as in mediator of DNA damage checkpoint protein 1 (MDC1). A body of evidence indicates that interaction between MDC1 and H2AX is the first step in which the site of the DSB is prepared for DNA damage signaling and repair (Falck et al., 2005, Stucki et al., 2005).

Chk1 and Chk2 are checkpoint kinases downstream of ATM and ATR and play a critical role in determining cellular responses to DNA damage. Chk1 is a serine/threonine kinase and is primarily responsible for initiating cell cycle arrest, allowing time for DNA repair and cell survival (Bartek and Lukas, 2001, Zhou and Sausville, 2003). After its activation, Chk1 phosphorylates the protein phosphatase Cdc25A, leading to its ubiquitination and degradation preventing cells from entering S phase (Mailand et al., 2000, Sørensen et al., 2003). Chk2 is also a serine/threonine kinase activated by phosphorylation by ATM following DSB. Once activated, the effect of Chk2 on the effector protein Cdc25A phosphatase is similar to that mediated by Chk 1 (Mailand et al., 2000, Falck et al., 2001). Fig. 2 illustrates the major pathways regulated by ATM, ATR, DNA-PK, and the DNA repair processes they are involved in.

When DNA damage occurs, cells use two major DNA DSB repair pathways: homologous recombination (HR) or non-homologous end joining (NHEJ), depending on the phase of the cell cycle and the condition of the DSB ends (Mimitou & Symington, 2009). HR provides accurate genetic recombination using a sister chromatid in S and G2 phases as a template, which is essential for the maintenance of genomic integrity. NHEJ is an error-prone method of directly ligating the DSB ends in the G0 and G1 phases, in which HR is not available. HR involves BRCA1, BRCA2 and Rad51 proteins while NHEJ involves Ku70/80, the DNA-PK, and DNA ligase IV. Other DNA repair mechanisms are also active during cell cycle or in response to DNA damage. Mismatch repair is very important during replication to remove mismatches, or small insertion or deletion loops, that are generated by faulty replication. Nucleotide-excision repair (NER), which is mainly responsible for repairing pyrimidine dimers, plays an important role during G1 phase to remove bulky lesions, such as those caused by ultraviolet irradiation. If left unrepaired during G1 phase, bulky DNA lesions can block DNA polymerases. Replication then proceeds by bypassing these lesions using specialized translesion synthesis (TLS) polymerases or template-switch mechanisms that use the newly synthesized sister chromatid as a template.