Abstract PL05-03: Exploiting cancer replication stress using pharmacological inhibitors of ATR and WEE1
Authors: O'Connor Mark J. 
Published: May 2016

Abstract
Replication stress is a hallmark of cancer (Macheret and Halazonetis, 2015) and as well as representing an important aspect of cancer aetiology (Bartkova et al., 2005; Gorgoulis et al., 2005) has the potential to be exploited by targeted cancer therapies (Lecona and Fernandez-Capetillo, 2014). A molecular definition of replication stress is the uncoupling of the DNA polymerase from the replisome helicase activity (Byun et al., 2005; Zeman and Cimprich, 2014). Factors that can induce replication stress include insufficiency in nucleotide pools or other replication factors, for example resulting from the abrogation of cell cycle control and premature entry into S-phase and DNA replication before the necessary resources for replication have been generated (Buisson et al., 2015). Cyclin E amplification and oncogenic drivers such KRAS mutations or MYC amplification can also increase replication stress through increased replication origin firing and the promotion of clashes between the replication and transcription processes and, in the case of MYC amplification through the generation of increased levels of reactive oxygen species and DNA damage (Jones et al., 2013; Rohban et al., 2014; Vafa et al., 2002).
Consistent with these causes, it can be seen why replication stress generated by DNA damaging chemotherapy will be greater in cancers than in normal cells, since cancers but not normal cells will also be associated with cell cycle checkpoint deregulation, oncogenic drivers, and higher levels of intrinsic DNA damage. However, there is still collateral damage in rapidly dividing normal cells such as gut epithelia, haematopoietic cells, and hair follicles. The goal for targeting cancer DNA damage response (DDR) dependencies associated with the replication stress response, is therefore to understand which DDR targets provide a potentially better therapeutic index than current standard of care chemotherapies and how best to select cancers that will demonstrate the greatest susceptibility to those DDR targeted agents.
The replication stress response initiated by RPA bound to ssDNA is regulated primarily by the Ataxia Telangiectasia and Rad3 Related serine/threonine kinase (ATR) and its effector kinase CHK1 that together control the firing of replication origins, the repair of damaged replication forks and the prevention of DNA double strand break formation (Cimprich and Cortez, 2008; Petermann et al., 2010; Buisson et al., 2015). The tyrosine kinase WEE1 also represents an important regulator of the replication stress response through the control of CDK1 and CDK2 activity and when WEE1 is inhibited, CDK1 and CDK2 de-regulation leads to increased replication origin firing, a decrease in nucleotide availability and an increase in DNA DSBs mediated through endonuclease activity (Beck et al, 2012). In addition, ATR, CHK1 and WEE1 inhibitors also have the potential to promote premature entry into mitosis, even in the presence of under-replicated DNA or DNA damage, thus promoting mitotic catastrophe and cancer cell death (Aarts et al., 2012; Do et al., 2013).
Here, data will be presented on the activity of both an ATR inhibitor (AZD6738) and a WEE1 inhibitor (AZD1775) in a number of preclinical in vitro and in vivo models. Initially, we assessed ATR inhibitor sensitivity in diffuse-large B-cell lymphoma (DLBCL) cancer models and identified an association of ATR inhibitor sensitivity with high levels of c-MYC protein, CDKN2A/B deletion and low BCL-6 expression and also demonstrated a strong correlation with the level of replication stress in these models. A similar trend in sensitivity in DLBCL was observed for the WEE1 inhibitor AZD1775 and the CHK1/2 inhibitor AZD7762, but with what appeared to be a greater potency compared to ATR inhibition. Investigation of both the cell cycle and cell death effects resulting from treatment with the ATR and WEE1 inhibitors in DLBCL models highlighted differences consistent with the greater potency of the WEE1 inhibitor and an assessment of in vivo activity further supported these findings. AZD1775 treatment of a larger panel of in vivo patient-derived explant (PDX) models of multiple tumour types demonstrated both the significant breadth and depth of the WEE1 inhibitor single agent activity. Moreover, we observed the potential for this activity to be enhanced further through combination with olaparib, a PARP inhibitor that induces S-phase DNA damage. Together, these data have led to the recent initiation of AZD1775 monotherapy as well as AZD1775/ olaparib combination clinical trials.
Citation Format: Mark J. O'Connor. Exploiting cancer replication stress using pharmacological inhibitors of ATR and WEE1. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2015 Nov 5-9; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2015;14(12 Suppl 2):Abstract nr PL05-03.