ABSTRACT
hMTH1 is an 8-oxodGTPase that prevents mis-incorporation of free oxidized nucleotides into genomic DNA. Base excision and mismatch repair pathways also restrict the accumulation of oxidized lesions in DNA by removing the mis-inserted 8-oxo-7,8-dihydro-2'-deoxyguanosines (8-oxodGs). In this study, we aimed to investigate the interplay between hMYH DNA glycosylase and hMTH1 for cancer cell survival by using mismatch repair defective T-cell acute lymphoblastic leukemia (T-ALL) cells. To this end, MYH and MTH1 were silenced individually or simultaneously using small hairpin RNAs. Increased sub-G1 population and apoptotic cells were observed upon concurrent depletion of both enzymes. Elevated cell death was consistent with cleaved caspase 3 accumulation in double knockdown cells. Importantly, overexpression of the nuclear isoform of hMYH could remove the G1 arrest and partially rescue the toxicity observed in hMTH1-depleted cells. In addition, expression profiles of human DNA glycosylases were generated using quantitative reverse transcriptase-PCR in MTH1 and/or MYH knockdown cells. NEIL1 DNA glycosylase, involved in repair of oxidized nucleosides, was found to be significantly downregulated as a cellular response to MTH1-MYH co-suppression. Overall, the results suggest that hMYH and hMTH1 functionally cooperate for effective repair and survival in mismatch repair defective T-ALL Jurkat A3 cells.
ABSTRACT
BACKGROUND: Previously, we showed cancer cells rely on the MTH1 protein to prevent incorporation of otherwise deadly oxidised nucleotides into DNA and we developed MTH1 inhibitors which selectively kill cancer cells. Recently, several new and potent inhibitors of MTH1 were demonstrated to be non-toxic to cancer cells, challenging the utility of MTH1 inhibition as a target for cancer treatment. MATERIAL AND METHODS: Human cancer cell lines were exposed in vitro to MTH1 inhibitors or depleted of MTH1 by siRNA or shRNA. 8-oxodG was measured by immunostaining and modified comet assay. Thermal Proteome profiling, proteomics, cellular thermal shift assays, kinase and CEREP panel were used for target engagement, mode of action and selectivity investigations of MTH1 inhibitors. Effect of MTH1 inhibition on tumour growth was explored in BRAF V600E-mutated malignant melanoma patient derived xenograft and human colon cancer SW480 and HCT116 xenograft models. RESULTS: Here, we demonstrate that recently described MTH1 inhibitors, which fail to kill cancer cells, also fail to introduce the toxic oxidized nucleotides into DNA. We also describe a new MTH1 inhibitor TH1579, (Karonudib), an analogue of TH588, which is a potent, selective MTH1 inhibitor with good oral availability and demonstrates excellent pharmacokinetic and anti-cancer properties in vivo. CONCLUSION: We demonstrate that in order to kill cancer cells MTH1 inhibitors must also introduce oxidized nucleotides into DNA. Furthermore, we describe TH1579 as a best-in-class MTH1 inhibitor, which we expect to be useful in order to further validate the MTH1 inhibitor concept.
