The most widely used anticancer agents prevent cell growth by damaging chromosomal DNA or inhibiting DNA replication (Kastan and Bartek, 2004
). Diverse types of DNA lesions and DNA structures trigger the activation of the phosphatydlinositol kinase-like kinases ATM and ATR. ATM is primarily activated by the double strand DNA breaks caused by ionizing radiation and radiomimetic drugs. ATR functions downstream of ATM in response to DNA damage (Jazayeri et al., 2006
), but is also activated independently of ATM by a wide range of agents that inhibit DNA replication and cause the accumulation of replication intermediates (Osborn et al., 2002
; Hurley and Bunz, 2007
; Cimprich and Cortez, 2008
). Replication inhibitors that can robustly activate ATR include antimetabolites that alter nucleotide metabolism and alkylating agents that cause DNA lesions that physically impede DNA replication forks. ATM and ATR directly phosphorylate over 700 downstream substrates that collectively control cell growth and survival (Matsuoka et al., 2007
Prominent among the regulatory proteins activated after DNA damage is the tumor suppressor p53, a transcription factor that is stabilized upon phosphorylation and thereby activated (Tibbetts et al., 1999
; Shiloh, 2006
). Genetic alterations that cause loss of p53 function - which occur in a large proportion of all human cancers - cause defective regulation of cell growth and death in response to DNA damage, and therefore present a potential obstacle to effective therapy (El-Deiry, 2003
; Meek, 2009
). Because the toxicity of most therapeutic agents to normal tissues limits the doses that can be safely administered to patients, strategies for selectively sensitizing p53-deficient cancer cells to existing anticancer drugs and radiation would have significant clinical impact.
Recent studies have demonstrated parallel interactions between upstream DNA damage signaling pathways and p53 that may be exploited to selectively impair coordinated cell cycle arrest (Chung and Bunz, 2010
) and improve therapeutic responses (Jiang et al., 2009
) in p53
-mutant cells. For example, targeted inhibition of ATM and its substrate Chk2 has been shown to increase the sensitivity of p53-/-
human cancer cells to the radiomimetic drug doxorubicin, while increasing the resistance of p53+/+
cells (Jiang et al., 2009
). It is currently unknown whether specific targeting of ATR might similarly increase the sensitivity of p53-deficient cells.
ATR is a particularly attractive target for combination therapies as it is robustly activated by many different types of drugs. Inhibiting ATR activity, either by RNAi-mediated knockdown of ATR
expression (Collis et al., 2003
) or by overexpression of a dominant-negative mutant ATR protein (Cliby et al., 1998
), has been shown to confer sensitivity to diverse anticancer agents, including ionizing radiation, methyl methanesulfonate and cisplatin.
To rigorously study the role of ATR in therapeutic responses, we generated a genetic model system wherein a human colorectal cancer cell line was engineered to harbor the hypomorphic mutation at the ATR
locus that causes ATR-Seckel syndrome (Hurley et al., 2007
). At the cellular level, the ATR-Seckel mutation causes aberrant splicing of the ATR
transcript and markedly decreased ATR expression (Alderton et al., 2004
; O'Driscoll et al., 2003
). Cancer cells homozygous for ATR-Seckel alleles (ATRS/S
) exhibit greatly reduced clonogenic survival in response to many commonly used anticancer agents, particularly to DNA crosslinking agents such as cisplatin (Wilsker and Bunz, 2007
). Decreased ATR expression also causes differential and highly reproducible sensitization to antimetabolites, ionizing radiation and radiomimetic drugs in this in vitro system (Hurley et al., 2007
; Wilsker and Bunz, 2007
). An important question that arises from these studies concerns the potential efficacy of anti-ATR therapy. Would inhibiting ATR preferentially sensitize cancer cells with loss of p53 function?
Recent studies in mice suggest that the effects of ATR inhibition on cell survival are antagonized by p53. In a mouse model of ATR-Seckel syndrome, the deficiency of ATR causes impaired DNA replication during embryogenesis and accelerated aging in adult mice (Murga et al., 2009
). Homozygous disruption of p53
in the ATR-Seckel background aggravates this aging phenotype. In a mosaic mouse model, the conditional disruption of ATR
in a p53
-mutant background causes the accumulation of DNA damage and tissue degradation (Ruzankina et al., 2009
). Together, these studies demonstrate that p53 functions to protect against the detrimental effects of ATR deficiency. In this study, we examined whether p53-mutant human cancer cells might be preferentially chemosensitized by genetic ATR inhibition. We show that RNAi-mediated knockdown of ATR preferentially sensitized p53-/-
cells to the effects of cisplatin, and that knockin of wild type p53
into the ATR-Seckel background suppressed apoptotic pathways, restored checkpoints and increased cisplatin resistance to the level exhibited by cells with wild type ATR
. These data support specific ATR inhibition as a therapeutic strategy to target p53-deficient tumors.