WEE1-inhibition and hypoxia : The impact of hypoxia and reoxygenation on the S-phase effects of the WEE1-inhibitor MK-1775

Abstract

The WEE1-inhibitor MK-1775 is currently in clinical trials as an anti-cancer drug. In response to DNA damage, human cells activate cell cycle checkpoints to temporarily halt cell cycle progression, and this helps facilitating DNA repair and is important to preserve genomic stability. It has been shown that inhibition of WEE1 leads to abrogation of the DNA-damage induced G2/M-checkpoint. Cancer cells often lack the p53-dependent G1/S-checkpoint, and may depend more on the G2/M-checkpoint for DNA damage repair than normal cells. The rationale behind using WEE1-inhibitors for cancer treatment is therefore that one can exploit this defective G1/S-checkpoint, in combination with G2/M-checkpoint abrogation, to make these cancer cells more sensitive towards DNA-damaging agents. However, recent studies have shown that in addition to regulating the G2/M-checkpoint, WEE1 also regulates normal S-phase progression in the absence of DNA-damaging agents. Previous work in our laboratory has shown that inhibiting WEE1 in normal S-phase causes DNA breakage and activates DNA damage signaling, and we believe that such S-phase damage also might contribute to cause cancer cell death following WEE1-inhibition. Hypoxia is a common trait of solid tumors, and it develops due to rapid growth of cancer cells and insufficient growth of new blood vessels in the tumor, resulting in inadequate delivery of oxygen to the tumor cells. Tumor hypoxia is known to cause resistance to radiation therapy and certain chemotherapeutic drugs. An important issue regarding new cancer therapeutic drugs is therefore to investigate how tumor hypoxia influences the efficacy of the drug. As MK-1775 is already being tested in ongoing clinical trials, it is important to find out how the response to the drug might be altered when cancer cells are exposed to hypoxic conditions. Furthermore, previous work in our laboratory has shown increased S-phase damage in response to inhibitors of a related kinase, CHK1, following hypoxic exposure. In addition, several studies have shown that severe hypoxia can activate DNA damage signaling and replication stalling in S-phase cells. In relations to this, we wanted to investigate the impact of hypoxia on the S-phase effects of the WEE1-inhibitor MK-1775. We performed experiments in U2OS osteosarcoma cells with several concentrations of MK-1775 at different levels of hypoxia. Our results showed no marked differences in the MK-1775-induced S-phase damage between normoxic cells and cells exposed to hypoxia. However, we found that the inhibitor is toxic to both hypoxic cells and in cells exposed to hypoxia followed by reoxygenation, demonstrating the potential for using MK-1775 for treatment of hypoxic tumors.