Abstract C59: Role of MTH1 (NUTD1) in cancer cell survival

• Published in: Molecular cancer therapeutics 2015-12, Vol.14 (12_Supplement_2), p.C59-C59
• Main Authors: Alwan, Husam, Eckersley, Kay, Goodwin, Louise, Lau, Alan, Jones, David, Kettle, Jason, Nissink, Willem M., Read, Jon, Scott, James S., Taylor, Benjamin J.M., Walker, Graeme E., Foote, Kevin M.
• Format: Article
• Language: English
• ISSN: 1535-7163; 1538-8514
• DOI: 10.1158/1535-7163.TARG-15-C59

Human mutT homolog MTH1 (also known as NUDT1) is a purine nucleoside triphosphatase which hydrolyses oxidised nucleotides (8-oxo-dGTP and 2-OH-dATP) into mono-phosphate forms to prevent these damaged bases from being incorporated into DNA. Recent studies have suggested a key role of MTH1 in the survival of cancer cells. It was hypothesized that in cancer cells with high levels of reactive oxygen species (ROS), small molecule inhibition or loss of MTH1 would lead to accumulation of oxidised nucleotides in DNA, increased genome instability and loss of cell viability. In order to validate MTH1 as a potential cancer therapeutic target we have developed additional potent small molecule inhibitors of MTH1 and generated siRNA knock-down and CRISPR-mediated knock-out cell lines. Using these tools we evaluated the impact of either MTH1 inhibition or loss of expression on cell viability, proliferation, induction of DNA damage and cell cycle arrest across multiple cell lines in vitro. While we have confirmed that the previous study compounds (TH588, TH287, (S)-crizotinib) are potent inhibitors of MTH1 and have broad cancer cell line growth inhibition activity, we were unable to demonstrate a corresponding induction of γH2AX, 53BP1 or phospho-ATM DNA damage response (DDR). In addition none of our MTH1 inhibitors were able to show significant growth inhibitory activity across cell lines panels or induce a DDR despite being able to potently engage MTH1 in cells as evidenced by cell thermal stability assay (CETSA). Due to the lack of DNA damage induction and inconsistent anti-proliferative activity between MTH1 inhibitors, we investigated the effects of loss of protein expression using additional, independent siRNA and CRISPR knockout cell lines. Despite potent and long lasting siRNA knockdown of MTH1 protein expression in U2OS cells, we did not observe significant differences in cell viability or induction in DNA damage when compared to control cell lines. Consistent with these findings we were also able to generate SW480 clones with complete knockout of all MTH1 alleles and this did not impair growth rates when compared to parental cells. Finally, to probe the specificity of MTH1 inhibitors we assessed their ability to impact the growth of these cells which do not express MTH1. We did not observe a differential response in U2OS cells with or without MTH1 siRNA or in the SW480 parental versus MTH1 knockout clones. Here we show through the use of novel small-molecule MTH