Activated RAS GTPase signalling is a critical driver of oncogenic transformation and malignant disease. Cellular models of RAS-dependent cancers have been used to identify experimental small molecules, such as SCH51344, but their molecular mechanism of action remains generally unknown. Here, using a chemical proteomic approach, we identify the target of SCH51344 as the human mutT homologue MTH1 (also known as NUDT1), a nucleotide pool sanitizing enzyme. Loss-of-function of MTH1 impaired growth of KRAS tumour cells, whereas MTH1 overexpression mitigated sensitivity towards SCH51344. Searching for more drug-like inhibitors, we identified the kinase inhibitor crizotinib as a nanomolar suppressor of MTH1 activity. Surprisingly, the clinically used (R)-enantiomer of the drug was inactive, whereas the (S)-enantiomer selectively inhibited MTH1 catalytic activity. Enzymatic assays, chemical proteomic profiling, kinome-wide activity surveys and MTH1 co-crystal structures of both enantiomers provide a rationale for this remarkable stereospecificity. Disruption of nucleotide pool homeostasis via MTH1 inhibition by (S)-crizotinib induced an increase in DNA single-strand breaks, activated DNA repair in human colon carcinoma cells, and effectively suppressed tumour growth in animal models. Our results propose (S)-crizotinib as an attractive chemical entity for further pre-clinical evaluation, and small-molecule inhibitors of MTH1 in general as a promising novel class of anticancer agents.
Subscribe to Journal
Get full journal access for 1 year
only $3.90 per issue
All prices are NET prices.
VAT will be added later in the checkout.
Rent or Buy article
Get time limited or full article access on ReadCube.
All prices are NET prices.
Protein Data Bank
Atomic coordinates for MTH1 in complex with (R)- and (S)-crizotinib have been deposited at the Protein Data Bank under accession codes 4c9w ((R)-crizotinib) and 4c9x ((S)-crizotinib), respectively. The protein interactions from this publication have been submitted to the IntAct database (http://www.ebi.ac.uk/intact/) and assigned the identifier EBI-9232460.
Pylayeva-Gupta, Y., Grabocka, E. & Bar-Sagi, D. RAS oncogenes: weaving a tumorigenic web. Nature Rev. Cancer 11, 761–774 (2011)
Parada, L. F., Tabin, C. J., Shih, C. & Weinberg, R. A. Human EJ bladder carcinoma oncogene is homologue of Harvey sarcoma virus ras gene. Nature 297, 474–478 (1982)
Der, C. J., Krontiris, T. G. & Cooper, G. M. Transforming genes of human bladder and lung carcinoma cell lines are homologous to the ras genes of Harvey and Kirsten sarcoma viruses. Proc. Natl Acad. Sci. USA 79, 3637–3640 (1982)
Dekker, F. J. et al. Small-molecule inhibition of APT1 affects Ras localization and signaling. Nature Chem. Biol. 6, 449–456 (2010)
Xu, J. et al. Inhibiting the palmitoylation/depalmitoylation cycle selectively reduces the growth of hematopoietic cells expressing oncogenic Nras. Blood 119, 1032–1035 (2012)
Zimmermann, G. et al. Small molecule inhibition of the KRAS–PDEδ interaction impairs oncogenic KRAS signalling. Nature 497, 638–642 (2013)
Yagoda, N. et al. RAS–RAF–MEK-dependent oxidative cell death involving voltage-dependent anion channels. Nature 447, 865–869 (2007)
Kumar, C. C. et al. SCH 51344 inhibits ras transformation by a novel mechanism. Cancer Res. 55, 5106–5117 (1995)
Walsh, A. B., Dhanasekaran, M., Bar-Sagi, D. & Kumar, C. C. SCH 51344-induced reversal of RAS-transformation is accompanied by the specific inhibition of the RAS and RAC-dependent cell morphology pathway. Oncogene 15, 2553–2560 (1997)
Rai, P. et al. Enhanced elimination of oxidized guanine nucleotides inhibits oncogenic RAS-induced DNA damage and premature senescence. Oncogene 30, 1489–1496 (2011)
Barbie, D. A. et al. Systematic RNA interference reveals that oncogenic KRAS-driven cancers require TBK1. Nature 462, 108–112 (2009)
Fujikawa, K. et al. The oxidized forms of dATP are substrates for the human MutT homologue, the hMTH1 protein. J. Biol. Chem. 274, 18201–18205 (1999)
Oka, S. et al. Two distinct pathways of cell death triggered by oxidative damage to nuclear and mitochondrial DNAs. EMBO J. 27, 421–432 (2008)
Yoshimura, D. et al. An oxidized purine nucleoside triphosphatase, MTH1, suppresses cell death caused by oxidative stress. J. Biol. Chem. 278, 37965–37973 (2003)
Svensson, L. M. et al. Crystal structure of human MTH1 and the 8-oxo-dGMP product complex. FEBS Lett. 585, 2617–2621 (2011)
Rai, P. et al. Continuous elimination of oxidized nucleotides is necessary to prevent rapid onset of cellular senescence. Proc. Natl Acad. Sci. USA 106, 169–174 (2009)
Fedorov, O. et al. Specific CLK inhibitors from a novel chemotype for regulation of alternative splicing. Chem. Biol. 18, 67–76 (2011)
Cui, J. J. et al. Structure based drug design of crizotinib (PF-02341066), a potent and selective dual inhibitor of mesenchymal–epithelial transition factor (c-MET) kinase and anaplastic lymphoma kinase (ALK). J. Med. Chem. 54, 6342–6363 (2011)
Zou, H. Y. et al. An orally available small-molecule inhibitor of c-Met, PF-2341066, exhibits cytoreductive antitumor efficacy through antiproliferative and antiangiogenic mechanisms. Cancer Res. 67, 4408–4417 (2007)
Camidge, D. R. et al. Activity and safety of crizotinib in patients with ALK-positive non-small-cell lung cancer: updated results from a phase 1 study. Lancet Oncol. 13, 1011–1019 (2012)
Kwak, E. L. et al. Anaplastic lymphoma kinase inhibition in non–small-cell lung cancer. N. Engl. J. Med. 363, 1693–1703 (2010)
Gerber, D. E. & Minna, J. D. ALK inhibition for non-small cell lung cancer: from discovery to therapy in record time. Cancer Cell 18, 548–551 (2010)
Butrynski, J. E. et al. Crizotinib in ALK-rearranged inflammatory myofibroblastic tumor. N. Engl. J. Med. 363, 1727–1733 (2010)
Martinez Molina, D. et al. Monitoring drug target engagement in cells and tissues using the cellular thermal shift assay. Science 341, 84–87 (2013)
Fabian, M. A. et al. A small molecule-kinase interaction map for clinical kinase inhibitors. Nature Biotechnol. 23, 329–336 (2005)
Zuber, J. et al. An integrated approach to dissecting oncogene addiction implicates a Myb-coordinated self-renewal program as essential for leukemia maintenance. Genes Dev. 25, 1628–1640 (2011)
Sakumi, K. et al. Ogg1 knockout-associated lung tumorigenesis and its suppression by Mth1 gene disruption. Cancer Res. 63, 902–905 (2003)
Speina, E. et al. Contribution of hMTH1 to the maintenance of 8-oxoguanine levels in lung DNA of non-small-cell lung cancer patients. J. Natl Cancer Inst. 97, 384–395 (2005)
Kennedy, C. H., Cueto, R., Belinsky, S. A., Lechner, J. F. & Pryor, W. A. Overexpression of hMTH1 mRNA: a molecular marker of oxidative stress in lung cancer cells. FEBS Lett. 429, 17–20 (1998)
Okamoto, K. et al. Overexpression of human mutT homologue gene messenger RNA in renal-cell carcinoma: evidence of persistent oxidative stress in cancer. Int. J. Cancer 65, 437–441 (1996)
Tsuzuki, T. et al. Spontaneous tumorigenesis in mice defective in the MTH1 gene encoding 8-oxo-dGTPase. Proc. Natl Acad. Sci. USA 98, 11456–11461 (2001)
Hutt, A. J. Chirality and pharmacokinetics: an area of neglected dimensionality? Drug Metabol. Drug Interact. 22, 79–112 (2007)
Aksoy, O. et al. The atypical E2F family member E2F7 couples the p53 and RB pathways during cellular senescence. Genes Dev. 26, 1546–1557 (2012)
The team at CeMM was supported by the Austrian Academy of Sciences, the GEN-AU initiative of the Austrian Federal Ministry for Science and Research, and “ASSET”, a project funded by the European Union within FP7. S.K., E.S. and J.M.E. are grateful for financial support from the SGC, a registered charity (number 1097737) that receives funds from the Canadian Institutes for Health Research, the Canada Foundation for Innovation, Genome Canada, GlaxoSmithKline, Pfizer, Eli Lilly, Takeda, AbbVie, the Novartis Research Foundation, Boehringer Ingelheim, the Ontario Ministry of Research and Innovation and the Wellcome Trust (Grant No. 092809/Z/10/Z). E.S. was supported by the European Union FP7 Grant No. 278568 “PRIMES”. T.H. was supported by the Torsten and Ragnar Söderberg Foundation, the Knut and Alice Wallenberg Foundation, the Swedish Research Council, the European Research Council and the Swedish Cancer Society. J.I.L. was supported by the European Union FP7 Career Integration Grant (PCIG11-GA-2012-321602) and an FWF Grant (P24766-B20). We are grateful to D. Treiber, J. Hunt, P. Gallant and G. Pallares from DiscoveRx for the KdELECT and scanMAX studies. We thank W. Lindner and N. Maier for chiral HPLC analyses, R. Lichtenecker for NMR measurements, A. C. Müller for the annotation of the MS/MS spectrum, M. Brehme for help with the figures, and H. Pickersgill and G. Vladimer for critically reading the manuscript. We are very grateful to the following colleagues for the respective reagents: S. Lowe for the miR30 vectors and pMLP-p53; R. Weinberg for pLKO.1 shMTH1 and pBABE-puro plasmids; W. Berger for SW480, DLD1 and SW620 cells; R. Oehler for PANC1 cells; W. Hahn and A. Gad for BJ-hTERT, BJ-hTERT-SV40T, BJ-hTERT-SV40T-KRASV12 cells, B. Vogelstein for p53−/− and p21−/− HCT116 cells; C. Gasche for LoVo and HCT15 cells; A. Nussenzweig for Atm wild type and Atm−/− mouse embryonic fibroblasts.
A patent has been filed with data generated in this manuscript where K.V.M.H. and G.S.-F. are listed as inventors.
Extended data figures and tables
a, Immunoblot showing a dose-dependent competition between MTH1 and free SCH51344 for the affinity probe (n = 1 per condition). b, Isothermal titration calorimetry results for SCH51344. Data were measured at 15 °C in 50 mM Tris-HCl pH 7.8, 150 mM NaCl. Errors given in the table represent the error of the nonlinear least squares fit to the experimental data (n = 1). c, Stable knockdown of MTH1 by shRNA reduces SW480 cell viability in a colony formation assay. Data are shown as mean ± s.e.m. and are based on three independent experiments (n = 3). Asterisks indicate significance by one-way ANOVA; NS, not significant. d, MTH1 overexpression decreases SW480 sensitivity towards SCH51344 as reflected by a shift in IC50 value (left). Data are shown as mean ± s.e.m. and are based on three independent experiments (n = 3). Similarly, MTH1 overexpression partially restores SW480 proliferation as compared to empty vector at a sub-lethal dose of SCH51344 (right). Notably, the overall proliferation rate is comparable for empty vector- and pBabe-MTH1-transduced cells. Bottom asterisks indicate significance between SCH51344-treated empty vector and pBabe-MTH1 cells as calculated by two-way ANOVA; DMSO-treated empty vector versus DMSO-treated pBabe-MTH1 is not significant except for the last data point. Data are shown as mean ± s.e.m. and are based on three independent experiments (n = 3).
a, Isothermal titration calorimetry results for both crizotinib enantiomers. Data were measured at 15 °C in 50 mM Tris-HCl pH 7.8, 150 mM NaCl. *Error given in the table represent the error of the nonlinear least squares fit to the experimental data (n = 1). b, Kd binding constants of both crizotinib enantiomers for the (R)-crizotinib cognate targets ALK, MET and ROS1. Data are shown as mean ± s.e.m. (n = 2). c, Pharmacologic c-MET kinase inhibition by a highly potent inhibitor (JNJ-38877605, c-MET IC50 = 4 nM) does not suppress growth of KRAS-mutated SW480 cells in contrast to the MTH1 inhibitors SCH51344 and (S)-crizotinib. Images are representative of three independent experiments (n = 3). d, MTH1 overexpression does not alter SW480 sensitivity towards (S)-crizotinib. Data are shown as mean ± s.e.m. and are based on three independent experiments (n = 3). e, (S)-Crizotinib target specificity analysis. Comparison of the probability of true interaction (SAINT) versus the magnitude of spectral count reduction upon competition with the free compound. MTH1 is clearly the only significant target identified by chemoproteomics as further supported by a high spectral count (disc diameter) and very low frequency of appearance in AP-MS negative control experiments found in the CRAPome database (colour code). f, In contrast, analysis of (R)-crizotinib targets reveals a large number of kinases as specific interactors of the clinical enantiomer. Data shown in panels e and f are based on two independent experiments for each condition (n = 2 per condition), and each replicate was analysed in two technical replicates. Source data
Screening of both (R)- and (S)-crizotinib against a panel of 456 recombinant human protein kinases indicates a marked difference in the ability of the two enantiomers to bind kinases. (R)-crizotinib has high affinity towards a large number of kinases, including its cognate targets MET, ALK and ROS1. Selectivity Score or S-score is a quantitative measure of compound selectivity. It is calculated by dividing the number of kinases that compounds bind to by the total number of distinct kinases tested, excluding mutant variants. S(35) = (number of non-mutant kinases with %Ctrl <35)/(number of non-mutant kinases tested). Source data
a, MTH1 crystal structure overview with (S)-crizotinib. (S)-Crizotinib is shown in cyan, MTH1 is in pink with light green alpha-helices and the loops covering the binding site in blue. b, As a with a molecular surface shown covering MTH1 apart from the binding site loops. c, MTH1 crystal structures with (R)- and (S)-crizotinib showing 2Fo − Fc electron density maps contoured at 1σ. (R)-Crizotinib is shown in yellow, MTH1 is in pink with light green alpha-helices and the loops covering the binding site in blue. d, As c except with (S)-crizotinib shown in cyan.
a, Crystallization of MTH1 complexes. b, Data collection and refinement statistics.
a, Quantification of 53BP1 foci formation in SW480 cells upon MTH1 inhibitor treatment. Concentrations are 5 µM for SCH51344 and 2 µM for each crizotinib enantiomer. Data are shown as mean ± s.d. (n = 3). Asterisks indicate significance by two-way ANOVA; NS, not significant. b, In line with results obtained for the MTH1 inhibitors SCH51344 and (S)-crizotinib, transient knockdown of MTH1 also induces formation of 53BP1 foci in SW480 cells. Images are representative and data are shown as mean ± s.d. based on three independent experiments (n = 3) (P < 0.05, t-test). c, Formation of 53BP1 foci correlates with increased 8-oxo-guanine staining in SW480 cells treated with the MTH1 inhibitors SCH51344 and (S)-, but not (R)-crizotinib. Images are representative of three independent experiments (n = 3). d, Modified OGG1-MUTYH comet assay. Treatment of U2OS cells with the MTH1 inhibitor (S)-crizotinib (5 µM) induces formation of DNA single-strand breaks due to activation of endogenous base excision repair. Addition of the 8-oxo-guanine- and 2-hydroxy-adenine-specific DNA glycosylases OGG1 and MUTYH leads to an increase in the mean tail moment (MTM) due to increased DNA cleavage at lesion sites. Data are shown as mean ± s.e.m. of three independent experiments (n = 3). e, The occurrence of DNA single-strand breaks induced by the MTH1 inhibitors SCH51344 and (S)-crizotinib is significantly decreased in SW480 cells overexpressing human MTH1 compared to empty vector transfected cells. Concentrations used are as in c. Numbers depict MTM ± s.d.; images are representative of three independent experiments (n = 3), statistical significance was determined using the Holm–Sidak method (P < 0.05) (n = 2).
a, Western blot evaluation of p53-shRNA knockdown efficiency. b, Viability curves from colony formation assays of SW480 cells expressing inducible non-targeting (shRen.713), or targeting anti-p53 shRNAs. Cells were cultured for 2 days either with or without doxycycline, plated in triplicate in six-well plates, and drugs added 24 h later. Colonies were stained with crystal violet and quantified using ultraviolet absorbance after dye solubilisation with ethanol. Data are shown as mean ± s.e.m. and are based on three independent experiments (n = 3).
a, Stable knockdown of MTH1 does not alter SW480 sensitivity towards ATM (KU55933) or ATR (VE821) kinase inhibition. Data are shown as mean ± s.e.m. and are based on three independent experiments (n = 3). b, Conversely, ATM status does not affect MTH1 inhibitor efficacy in immortalized mouse embryonic fibroblasts. Data are shown as mean ± s.e.m. and are based on three independent experiments (n = 3). c, As observed for SW480, loss of p53 does not impair the sensitivity of KRAS-mutant HCT116 towards MTH1 inhibitors; however, p21−/− cells are more sensitive, in particular to the more potent MTH1 inhibitor (S)-crizotinib (top). Data are shown as mean ± s.e.m. and are based on three independent experiments (n = 3). WT, wild type. Similarly, BRCA2 function does not alter MTH1 inhibitor sensitivity of VC-8 cells (bottom). Data are shown as mean ± s.e.m. and are based on three independent experiments (n = 3).
a, BJ cells transformed by KRASV12 or SV40T are more sensitive to the MTH1 inhibitors SCH51344 and (S)-crizotinib than wild type fibroblasts or cells immortalized by telomerase expression. Data are shown as mean ± s.e.m. for three independent experiments (n = 3). b, (S)-Crizotinib does not exhibit any increased unspecific cytotoxicity compared to (R)-crizotinib. In contrast, the (R)-enantiomer significantly impairs the growth of untransformed BJ skin fibroblasts at low micromolar concentrations in a colony formation assay. Compounds were added 24 h after seeding the cells and plates were incubated for 10 days, washed, fixed, and stained with crystal violet. Images are representative of two independent experiments (n = 2). c, IC50 values for MTH1 inhibitors tested against a cancer cell line panel.
a, Mouse haematology and liver/heart/kidney parameters comparing treatment versus controls. SCID mice (n = 8 per group) were subcutaneously administered vehicle or (S)-crizotinib (25 mg per kg) for 35 days. Blood samples were obtained by orbital bleeding (under anaesthesia); blood parameters were analysed using whole blood and ASAT, ALAT and creatinine were analysed in EDTA-collected plasma by the Karolinska Universitetslaboratoriet, Clinical Chemistry. The mean values of white blood cells (WBC), red blood cells (RBC), neutrophils, lymphocytes, monocytes, mean corpuscular volume (MCV), mean cell haemoglobin (MCH), mean cell haemoglobin concentration (MCHC) from the different groups are presented in the table. The results did not show any significant differences between control and treated groups apart from a minor change in MCHC. b, Effect of (R)-crizotinib (50 mg per kg, orally, daily), (S)-crizotinib (50 mg per kg, orally, daily) or vehicle on tumour volume at day 26 in SW480 xenograft mice. Individual data are shown, n = 7–8 animals per group. Statistical analysis performed by two-way repeat measurement ANOVA, followed by Sidak’s multiple comparison. c, Effect of treatment on body weight. Data show mean ± s.e.m. d, Meta-analysis of Oncomine data. MTH1 expression strongly correlates with upregulated RAS, which is also reflected by the fact that cancers with high prevalence of RAS mutations such as lung and colon carcinoma express higher levels of MTH1 than other unrelated cancer types.
About this article
Cite this article
Huber, K., Salah, E., Radic, B. et al. Stereospecific targeting of MTH1 by (S)-crizotinib as an anticancer strategy. Nature 508, 222–227 (2014). https://doi.org/10.1038/nature13194
International Journal of Molecular Sciences (2020)
TH1579, MTH1 inhibitor, delays tumour growth and inhibits metastases development in osteosarcoma model
A profile of 8-oxo-dGTPase activities in the NCI-60 human cancer panel: Meta-analytic insight into the regulation and role of MTH1 (NUDT1) gene expression in carcinogenesis
Free Radical Biology and Medicine (2020)
In silico Druggability Assessment of the NUDIX Hydrolase Protein Family as a Workflow for Target Prioritization
Frontiers in Chemistry (2020)
Thoracic Surgery Clinics (2020)