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Stereospecific targeting of MTH1 by (S)-crizotinib as an anticancer strategy.

Huber KV, Salah E, Radic B, Gridling M, Elkins JM, Stukalov A, Jemth AS, Göktürk C, Sanjiv K, Strömberg K, Pham T, Berglund UW, Colinge J, Bennett KL, Loizou JI, Helleday T, Knapp S, Superti-Furga G - Nature (2014)

Bottom Line: 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.

View Article: PubMed Central - PubMed

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

ABSTRACT
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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Confirmation of MTH1 as the main cellular target of SCH51344a, Immunoblot showing a dose-dependent competition between MTH1 and free SCH51344 for the affinity probe (n = 1/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. Error given in the table represent the error of the nonlinear least squares fit to the experimental data (n = 1). c, Stable knock-down of MTH1 by shRNA reduces SW480 cell viability in a colony formation assay. Data are shown as mean ± SEM 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 toward SCH51344 as reflected by a shift in IC50 value (left). Data are shown as mean ± SEM 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 ± SEM and are based on three independent experiments (n = 3).
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Figure 5: Confirmation of MTH1 as the main cellular target of SCH51344a, Immunoblot showing a dose-dependent competition between MTH1 and free SCH51344 for the affinity probe (n = 1/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. Error given in the table represent the error of the nonlinear least squares fit to the experimental data (n = 1). c, Stable knock-down of MTH1 by shRNA reduces SW480 cell viability in a colony formation assay. Data are shown as mean ± SEM 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 toward SCH51344 as reflected by a shift in IC50 value (left). Data are shown as mean ± SEM 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 ± SEM and are based on three independent experiments (n = 3).

Mentions: We set out to identify the cellular targets of SCH51344 using a chemical proteomic strategy (Fig. 1a). We generated a SCH51344 affinity probe (Fig. 1b) which we incubated with lysates of KRAS-positive SW480 cells, which are sensitive to SCH51344, and analysed the binding proteins by mass spectrometry. High affinity binders were discriminated against highly abundant low affinity proteins by competition with the free unmodified compound. Bioinformatic analysis revealed the human 7,8-dihydro-8-oxoguanine triphosphatase MTH1 (also known as NUDT1) and adenosine kinase (ADK) as the primary cellular targets of SCH51344 (Fig. 1c). MTH1 has been implicated in aiding RAS-transformed cells to overcome oncogene-induced senescence by preventing reactive oxygen species (ROS)-induced DNA damage10. On the contrary, little was known about the role of ADK in malignant disease but in line with published RNAi data11 we did not observe any growth impairment of SCH51344-sensitive8 PANC1 human pancreatic carcinoma cells upon treatment with the ADK inhibitor ABT-702 (data not shown). We focussed on MTH1 as the most likely relevant target of SCH51344. Having confirmed the binding of SCH51344 to MTH1 in both SW480 and DLD1 cells by immunoblot (Extended Data Fig. 1a), we used isothermal titration calorimetry (ITC) to determine a Kd value of 49 nM for SCH51344 (Fig. 1d, Extended Data Fig. 1b). MTH1 is a homologue of the bacterial mutT, a nucleotide pool sanitising enzyme which cleaves oxidised nucleotides such as 8-oxo-deoxyguanosinetri-phosphate (8-oxo-dGTP) thereby converting the triphosphates into the corresponding mono-phosphates12. The hydrolysis reaction assures that the oxidised nucleotides can no longer be recognised by DNA polymerase, preventing the mispairing of bases during replication and transversion mutations13,14. To investigate the effect of SCH51344 on MTH1 catalytic activity we monitored the production of pyrophosphate (PPi) as a result of nucleotide triphosphate hydrolysis15. We determined IC50 values of 215 nM, 410 nM, and 675 nM for SCH51344 against the MTH1 substrates dGTP, 8-oxo-dGTP, and 2-OH-dATP, confirming a direct effect of SCH51344 on MTH1 catalytic activity (Fig. 1e). To validate MTH1 as the causal target for the antiproliferative effects of SCH51344, we transfected human SW480 and DLD1 cells with MTH1-siRNA which impaired colony formation (Fig. 1f). Stable knockdown using lentiviral shRNAs16 phenocopied results obtained using the inhibitor (Extended Data Fig. 1c). Conversely, overexpression of MTH110 reduced sensitivity of SW480 cells against SCH51344 (Fig. 1g, Extended Data Fig. 1d), mechanistically corroborating the evidence that MTH1 is the main cellular target of SCH51344.


Stereospecific targeting of MTH1 by (S)-crizotinib as an anticancer strategy.

Huber KV, Salah E, Radic B, Gridling M, Elkins JM, Stukalov A, Jemth AS, Göktürk C, Sanjiv K, Strömberg K, Pham T, Berglund UW, Colinge J, Bennett KL, Loizou JI, Helleday T, Knapp S, Superti-Furga G - Nature (2014)

Confirmation of MTH1 as the main cellular target of SCH51344a, Immunoblot showing a dose-dependent competition between MTH1 and free SCH51344 for the affinity probe (n = 1/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. Error given in the table represent the error of the nonlinear least squares fit to the experimental data (n = 1). c, Stable knock-down of MTH1 by shRNA reduces SW480 cell viability in a colony formation assay. Data are shown as mean ± SEM 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 toward SCH51344 as reflected by a shift in IC50 value (left). Data are shown as mean ± SEM 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 ± SEM and are based on three independent experiments (n = 3).
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Figure 5: Confirmation of MTH1 as the main cellular target of SCH51344a, Immunoblot showing a dose-dependent competition between MTH1 and free SCH51344 for the affinity probe (n = 1/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. Error given in the table represent the error of the nonlinear least squares fit to the experimental data (n = 1). c, Stable knock-down of MTH1 by shRNA reduces SW480 cell viability in a colony formation assay. Data are shown as mean ± SEM 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 toward SCH51344 as reflected by a shift in IC50 value (left). Data are shown as mean ± SEM 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 ± SEM and are based on three independent experiments (n = 3).
Mentions: We set out to identify the cellular targets of SCH51344 using a chemical proteomic strategy (Fig. 1a). We generated a SCH51344 affinity probe (Fig. 1b) which we incubated with lysates of KRAS-positive SW480 cells, which are sensitive to SCH51344, and analysed the binding proteins by mass spectrometry. High affinity binders were discriminated against highly abundant low affinity proteins by competition with the free unmodified compound. Bioinformatic analysis revealed the human 7,8-dihydro-8-oxoguanine triphosphatase MTH1 (also known as NUDT1) and adenosine kinase (ADK) as the primary cellular targets of SCH51344 (Fig. 1c). MTH1 has been implicated in aiding RAS-transformed cells to overcome oncogene-induced senescence by preventing reactive oxygen species (ROS)-induced DNA damage10. On the contrary, little was known about the role of ADK in malignant disease but in line with published RNAi data11 we did not observe any growth impairment of SCH51344-sensitive8 PANC1 human pancreatic carcinoma cells upon treatment with the ADK inhibitor ABT-702 (data not shown). We focussed on MTH1 as the most likely relevant target of SCH51344. Having confirmed the binding of SCH51344 to MTH1 in both SW480 and DLD1 cells by immunoblot (Extended Data Fig. 1a), we used isothermal titration calorimetry (ITC) to determine a Kd value of 49 nM for SCH51344 (Fig. 1d, Extended Data Fig. 1b). MTH1 is a homologue of the bacterial mutT, a nucleotide pool sanitising enzyme which cleaves oxidised nucleotides such as 8-oxo-deoxyguanosinetri-phosphate (8-oxo-dGTP) thereby converting the triphosphates into the corresponding mono-phosphates12. The hydrolysis reaction assures that the oxidised nucleotides can no longer be recognised by DNA polymerase, preventing the mispairing of bases during replication and transversion mutations13,14. To investigate the effect of SCH51344 on MTH1 catalytic activity we monitored the production of pyrophosphate (PPi) as a result of nucleotide triphosphate hydrolysis15. We determined IC50 values of 215 nM, 410 nM, and 675 nM for SCH51344 against the MTH1 substrates dGTP, 8-oxo-dGTP, and 2-OH-dATP, confirming a direct effect of SCH51344 on MTH1 catalytic activity (Fig. 1e). To validate MTH1 as the causal target for the antiproliferative effects of SCH51344, we transfected human SW480 and DLD1 cells with MTH1-siRNA which impaired colony formation (Fig. 1f). Stable knockdown using lentiviral shRNAs16 phenocopied results obtained using the inhibitor (Extended Data Fig. 1c). Conversely, overexpression of MTH110 reduced sensitivity of SW480 cells against SCH51344 (Fig. 1g, Extended Data Fig. 1d), mechanistically corroborating the evidence that MTH1 is the main cellular target of SCH51344.

Bottom Line: 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.

View Article: PubMed Central - PubMed

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

ABSTRACT
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.

Show MeSH
Related in: MedlinePlus