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.

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Data deposits

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.


  1. 1.

    , & RAS oncogenes: weaving a tumorigenic web. Nature Rev. Cancer 11, 761–774 (2011)

  2. 2.

    , , & Human EJ bladder carcinoma oncogene is homologue of Harvey sarcoma virus ras gene. Nature 297, 474–478 (1982)

  3. 3.

    , & 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)

  4. 4.

    et al. Small-molecule inhibition of APT1 affects Ras localization and signaling. Nature Chem. Biol. 6, 449–456 (2010)

  5. 5.

    et al. Inhibiting the palmitoylation/depalmitoylation cycle selectively reduces the growth of hematopoietic cells expressing oncogenic Nras. Blood 119, 1032–1035 (2012)

  6. 6.

    et al. Small molecule inhibition of the KRAS–PDEδ interaction impairs oncogenic KRAS signalling. Nature 497, 638–642 (2013)

  7. 7.

    et al. RAS–RAF–MEK-dependent oxidative cell death involving voltage-dependent anion channels. Nature 447, 865–869 (2007)

  8. 8.

    et al. SCH 51344 inhibits ras transformation by a novel mechanism. Cancer Res. 55, 5106–5117 (1995)

  9. 9.

    , , & 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)

  10. 10.

    et al. Enhanced elimination of oxidized guanine nucleotides inhibits oncogenic RAS-induced DNA damage and premature senescence. Oncogene 30, 1489–1496 (2011)

  11. 11.

    et al. Systematic RNA interference reveals that oncogenic KRAS-driven cancers require TBK1. Nature 462, 108–112 (2009)

  12. 12.

    et al. The oxidized forms of dATP are substrates for the human MutT homologue, the hMTH1 protein. J. Biol. Chem. 274, 18201–18205 (1999)

  13. 13.

    et al. Two distinct pathways of cell death triggered by oxidative damage to nuclear and mitochondrial DNAs. EMBO J. 27, 421–432 (2008)

  14. 14.

    et al. An oxidized purine nucleoside triphosphatase, MTH1, suppresses cell death caused by oxidative stress. J. Biol. Chem. 278, 37965–37973 (2003)

  15. 15.

    et al. Crystal structure of human MTH1 and the 8-oxo-dGMP product complex. FEBS Lett. 585, 2617–2621 (2011)

  16. 16.

    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)

  17. 17.

    et al. Specific CLK inhibitors from a novel chemotype for regulation of alternative splicing. Chem. Biol. 18, 67–76 (2011)

  18. 18.

    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)

  19. 19.

    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)

  20. 20.

    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)

  21. 21.

    et al. Anaplastic lymphoma kinase inhibition in non–small-cell lung cancer. N. Engl. J. Med. 363, 1693–1703 (2010)

  22. 22.

    & ALK inhibition for non-small cell lung cancer: from discovery to therapy in record time. Cancer Cell 18, 548–551 (2010)

  23. 23.

    et al. Crizotinib in ALK-rearranged inflammatory myofibroblastic tumor. N. Engl. J. Med. 363, 1727–1733 (2010)

  24. 24.

    et al. Monitoring drug target engagement in cells and tissues using the cellular thermal shift assay. Science 341, 84–87 (2013)

  25. 25.

    et al. A small molecule-kinase interaction map for clinical kinase inhibitors. Nature Biotechnol. 23, 329–336 (2005)

  26. 26.

    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)

  27. 27.

    et al. Ogg1 knockout-associated lung tumorigenesis and its suppression by Mth1 gene disruption. Cancer Res. 63, 902–905 (2003)

  28. 28.

    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)

  29. 29.

    , , , & Overexpression of hMTH1 mRNA: a molecular marker of oxidative stress in lung cancer cells. FEBS Lett. 429, 17–20 (1998)

  30. 30.

    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)

  31. 31.

    et al. Spontaneous tumorigenesis in mice defective in the MTH1 gene encoding 8-oxo-dGTPase. Proc. Natl Acad. Sci. USA 98, 11456–11461 (2001)

  32. 32.

    Chirality and pharmacokinetics: an area of neglected dimensionality? Drug Metabol. Drug Interact. 22, 79–112 (2007)

  33. 33.

    et al. The atypical E2F family member E2F7 couples the p53 and RB pathways during cellular senescence. Genes Dev. 26, 1546–1557 (2012)

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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.

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  1. CeMM Research Center for Molecular Medicine of the Austrian Academy of Sciences, 1090 Vienna, Austria

    • Kilian V. M. Huber
    • , Branka Radic
    • , Manuela Gridling
    • , Alexey Stukalov
    • , Jacques Colinge
    • , Keiryn L. Bennett
    • , Joanna I. Loizou
    •  & Giulio Superti-Furga
  2. Nuffield Department of Clinical Medicine, Structural Genomics Consortium, University of Oxford, Old Road Campus Research Building, Roosevelt Drive, Oxford OX3 7DQ, UK

    • Eidarus Salah
    • , Jonathan M. Elkins
    •  & Stefan Knapp
  3. Science for Life Laboratory, Division of Translational Medicine and Chemical Biology, Department of Medical Biochemistry and Biophysics, Karolinska Institutet, 17121 Stockholm, Sweden

    • Ann-Sofie Jemth
    • , Camilla Göktürk
    • , Kumar Sanjiv
    • , Kia Strömberg
    • , Therese Pham
    • , Ulrika Warpman Berglund
    •  & Thomas Helleday


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K.V.M.H., E.S., B.R., M.G., J. M.E., J.I.L., A.-S.J., K.S. performed experiments. K.V.M.H. and G.S.-F. conceived the study. K.V.M.H., J.I.L., U.W.B., T.H., S.K. and G.S.-F. designed experiments. A.S., K.L.B. and J.C. performed mass spectrometry and bioinformatic data analysis. C.G., K.S., T.P. and U.W.B. performed animal experiments. K.V.M.H., S.K. and G.S.-F. wrote the manuscript. All authors contributed to the discussion of results and participated in manuscript preparation.

Competing interests

A patent has been filed with data generated in this manuscript where K.V.M.H. and G.S.-F. are listed as inventors.

Corresponding author

Correspondence to Giulio Superti-Furga.

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