Exploiting universal cancer vulnerabilities has been used as an approach for developing targeted therapies. In this issue of Cancer Research, Rudd and colleagues show that the dual-functioning inhibitor TH588 potentiates the accumulation of reactive oxygen species during mitosis in cancer by disturbing mitotic progression and simultaneously inhibiting the hydrolysis of 8oxodGTP. This leads to increased incorporation of 8oxodG into the DNA during mitotic replication and increased toxicity. Understanding the mechanism of this inhibitor lays the groundwork for identifying cancer targets.
See related article by Rudd et al., p. 3530
In the early 1990s, Miller and colleagues (for a review, see ref. 1) described the 8-oxo-7,8-dihydrodeoxyguanine (GO) system of Escherichia coli (E. coli) for processing of oxidative DNA damage, namely, 8-oxoguanine (8oxoG). Removal of the 8oxoG and lesion bypass are key aspects of the GO system as is sanitization of the oxidized dNTP pool by the MutT protein. This system is highly conserved from E. coli to humans, and the oxidized purine diphosphatase, MTH1, which is the human homolog of MutT, also sanitizes the pool in human cells.
Central role for mitotic DNA synthesis in drug responses
TH287 and TH588 are first-in-class nudix hydrolase inhibitors that bind to the active site of MTH1 and inhibit its activity (2). This is important because cancer cells overexpress MTH1, which is likely to prevent the incorporation of oxidized nucleotides that arise during replication stress. Inhibition of MTH1 is suggested to permit the misincorporation of oxidized dNTPs into DNA, resulting in massive DNA damage and leading to cell death. The best-in-class drug derived from TH588 is TH1579 (karonudib) and is currently in clinical trials for patients with leukemia. Recent work provides evidence that TH588 targets the mitotic spindle and not MTH1 (3). Specifically, TH588 binds to the colchicine binding site of β-tubulin and blocks microtubule assembly, leading to mitotic arrest. However, cell death after treatment with TH588 is shown to be independent of MTH1 expression.
In this issue of Cancer Research, Rudd and colleagues propose that TH588 has dual functionality that is necessary for cell death (4). They suggest that inhibition of both microtubule polymerization and MTH1 enzymatic activity results in incorporation of 8oxoG into DNA during mitotic DNA synthesis, leading to cell death (4).
Specifically, TH588 interferes with microtubule polymerization, leading to mitotic arrest. During mitotic arrest, there is a buildup of reactive oxygen species (ROS), resulting in the accumulation of 8oxodGTP. In fact, it has been shown that ROS levels peak during mitosis (5). High levels of ROS could lead to increased incorporation of oxidized dNTPs and also to increased oxidative damage, both of which could result in increased levels of 8oxoG in DNA. Mitotic DNA synthesis is triggered upon mitotic arrest. Mitotic DNA synthesis is a specialized form of DNA synthesis that takes place at difficult to replicate common fragile sites (CFS) in the genome and initiates as cells enter prophase (6). Specifically, MUS81 nuclease activity promotes POLD3-dependent DNA synthesis at CFS, and perhaps other difficult to replicate sites during mitosis. Rudd and colleagues propose that inhibition of the MTH1 hydrolase activity by TH588 results in the misincorporation of 8oxoG into DNA during mitotic DNA synthesis, which leads to cell death (4). Unfortunately, the authors did not demonstrate that cell death is dependent upon the presence of MTH1. In fact, previous work by others showed that TH588 synergized with an inhibitor of PLK1 to kill cells, but that cell death was not dependent upon expression of MTH1 (3). Strikingly, inhibition of PLK1 was previously shown to also inhibit mitotic DNA synthesis (6). Therefore, inhibition of the enzymatic activity of MTH1 specifically during mitotic DNA synthesis could be critically important for cell death, as suggested by Rudd and colleagues. Interestingly, the cytotoxic effect of TH588 was shown to be enhanced in the presence of a mutator variant of POLD3, which is proposed to have better tolerance for the incorporation of 8oxoG. However, future studies on the ability of POLD3 to incorporate oxidized dNTPs will be important.
8oxoG may activate the MAPK pathway
8oxoG can also activate additional signaling pathways as has been previously proposed (7). This oxidized base has been shown to activate Ras and the downstream MAPK pathway, which may contribute to the cellular response to increased 8-oxoG incorporation. The MAPK pathway plays an important role during mitosis. The activation of ERK, which is downstream of Ras, has been shown to enhance microtubule depolymerization and to act as a checkpoint for metaphase spindle formation. Thus, the potential activation of ERK through an 8oxoG-associated process could also lead to mitotic arrest. Probing for the activated MAPK pathway might be an important mechanistic aspect for understanding the cytotoxicity of TH588.
Transcription and ROS
What are the biological implications of the rising levels of ROS during mitosis? On one hand, this could lead to DNA, RNA, and protein oxidation, which would not be good for the cell. On the other hand, one wonders if increased ROS is important for the cell once it exits mitosis, perhaps for a rapid restart of gene expression. Specifically, 8-oxoG, which is recognized by OGG1, has been suggested to act in an epigenetic manner to promote transcription.
In fact, mitotic transcription (8), occurs at low, perhaps baseline levels, and preferentially in genes that need to be expressed just after mitosis to support critical cellular functions. Interestingly, OGG1 is localized to condensed chromatin from prometaphase through telophase (9). OGG1 is thought to function in transcription (10) by binding to 8oxoG and facilitating recruitment of transcription factors and also by altering the structure of DNA, that is, G-quadraplex structures, permitting access to them by apurinic/apyrimidinic exonuclease 1 (APE1) and concomitant gene expression. Significantly increased 8oxoG incorporation into DNA, as would occur upon inhibition of MTH1, would be predicted to lead to increased levels of OGG1-facilitated transcription. Mitotic replication-transcription conflicts resulting from high levels of transcription could ultimately result in cell death. Probing this by treatment of cells with the OGG1 inhibitor (TH5487) could be informative as to whether OGG1 plays an important role in mitotic transcription and whether defective interaction of OGG1 alters mitotic outcomes.
Treatment of hypoxic tumors
Finally, this dual inhibitor may present an opportunity to target hypoxic tumors. These tumors are hard to treat and exhibit resistance to radiotherapy due to the reduced ability to create ROS in the absence of oxygen. Thus, using TH588 to potentiate the production of ROS during mitosis may be an effective mechanism for the treatment of hypoxic tumors and could be used as an alternative to radiotherapy.
Disclosure of Potential Conflicts of Interest
No potential conflicts of interest were disclosed.