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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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(S)-Crizotinib is a selective MTH1 inhibitor with in vivo anticancer activitya, The MTH1 inhibitors SCH51344 (5 μM) and (S)-crizotinib (2 μM), but not (R)-crizotinib (2 μM), induce DNA damage as indicated by an increase in 53BP1 foci and ATM autophosphorylation. Images are representative for three independent experiments (n = 3). b, Comet assay. Similar to MTH1 gene silencing both SCH51344 (5 μM) and (S)-crizotinib (2 μM), but not (R)-crizotinib (2 μM), induce DNA single strand breaks (MTM, mean tail moment). H2O2 was used as positive control (150 μM, 10 min). Images are representative for three independent experiments (n = 3), data are shown as mean ± SD. c, MTH1 overexpression reduces the number of DNA single strand breaks induced by SCH51344 and (S)-crizotinib. Compound concentrations are as in b. d, Results from SW480 mouse xenograft study. Effect on tumour growth following 35 days treatment with the MTH1 inhibitor (S)-crizotinib (25mg/kg q.d., s.c. daily, data are shown as mean ± SEM, n = 8/group). e, (S)-Crizotinib, but not (R)-crizotinib, impairs tumour growth in an SW480 colon carcinoma xenograft model (50mg/kg p.o., q.d.) Data show mean ± SEM, n=7-8 animals/group. Statistical analysis performed by 2-way repeat measurement ANOVA, Sidak’s multiple comparison; * p < 0.05 (S)-crizotinib vs control; † p < 0.05 (S)-crizotinib vs (R)-crizotinib. Images depict representative tumours for each treatment group (C, control). f, Proposed mechanism for MTH1-inhibitor-induced cancer cell death.
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Figure 4: (S)-Crizotinib is a selective MTH1 inhibitor with in vivo anticancer activitya, The MTH1 inhibitors SCH51344 (5 μM) and (S)-crizotinib (2 μM), but not (R)-crizotinib (2 μM), induce DNA damage as indicated by an increase in 53BP1 foci and ATM autophosphorylation. Images are representative for three independent experiments (n = 3). b, Comet assay. Similar to MTH1 gene silencing both SCH51344 (5 μM) and (S)-crizotinib (2 μM), but not (R)-crizotinib (2 μM), induce DNA single strand breaks (MTM, mean tail moment). H2O2 was used as positive control (150 μM, 10 min). Images are representative for three independent experiments (n = 3), data are shown as mean ± SD. c, MTH1 overexpression reduces the number of DNA single strand breaks induced by SCH51344 and (S)-crizotinib. Compound concentrations are as in b. d, Results from SW480 mouse xenograft study. Effect on tumour growth following 35 days treatment with the MTH1 inhibitor (S)-crizotinib (25mg/kg q.d., s.c. daily, data are shown as mean ± SEM, n = 8/group). e, (S)-Crizotinib, but not (R)-crizotinib, impairs tumour growth in an SW480 colon carcinoma xenograft model (50mg/kg p.o., q.d.) Data show mean ± SEM, n=7-8 animals/group. Statistical analysis performed by 2-way repeat measurement ANOVA, Sidak’s multiple comparison; * p < 0.05 (S)-crizotinib vs control; † p < 0.05 (S)-crizotinib vs (R)-crizotinib. Images depict representative tumours for each treatment group (C, control). f, Proposed mechanism for MTH1-inhibitor-induced cancer cell death.

Mentions: Since MTH1 is thought to prevent incorporation of oxidised nucleotides into DNA, we reasoned that our new MTH1 inhibitors should increase the content of genomic 8-oxoguanine, and thus induce DNA damage. Immunofluorescence staining for both 53BP1 and autophosphorylated ATM, specific markers for DNA damage, was increased in SW480 cells treated with MTH1 inhibitors (Fig. 4a, Extended Data Fig. 6a). 53BP1 foci, which we also observed in cells transfected with MTH1-siRNA, were enriched in nuclei of cells with higher levels of 8-oxoguanine due to increased genomic incorporation (Extended Data Fig. 6b, c). We also tried to quantify the oxidized nucleotides by HPLC-MS, however, due to high experimental background we failed to obtain reliable results. Since accumulation of 8-oxoguanine should activate base excision repair (BER)16 and induce DNA single strand breaks, we tested our inhibitors in an alkaline comet assay. Both (S)-crizotinib as well as SCH51344, but not (R)-crizotinib, yielded a significant tail moment, similar to cells transfected with MTH1-siRNA (Fig. 4b). Addition of the purified 8-oxoguanine- or 2-hydroxy-adenine-specific DNA glycosylases OGG1 and MUTYH, increased tail moments for (S)-crizotinib dramatically, providing evidence for strong accumulation of these lesions upon inhibitor treatment (Extended Data Fig. 6d). MTH1 overexpression significantly reduced the number of DNA single strand breaks induced by (S)-crizotinib as well as SCH51344, but not by H2O2 (Fig. 4c, Extended Data Fig. 6e) providing evidence for MTH1 being the functionally relevant target. To explore the role of p53 in the cellular response to MTH1 suppression16 we created a SW480 Tet-on system26 allowing for the inducible expression of p53 shRNAs and treated the cells with our inhibitors (Extended Data Fig. 7) which indicated a p53-independent mode of action. Treatment of SW480 cells expressing anti-MTH1 shRNA with the ATM- and ATR-inhibitors KU55933 and VE821, respectively, also did not display any differential effects (Extended Data Fig. 8). Similarly, ATM−/− MEFs were equally sensitive to MTH1 inhibitors as their ATM-proficient counterparts. Investigating cell lines bearing additional mutations in DNA repair genes we found that HCT116 deficient for p21 were particularly sensitive to (S)-crizotinib. We tested our inhibitors in BJ skin fibroblasts that were either wild type, immortalized by hTERT, or transformed by SV40T and/or mutant KRAS. Both SCH51344 and (S)-crizotinib exhibited highest toxicity toward the SV40T and KRAS-V12 cells (Extended Data Fig. 9). Importantly, when we treated wild type BJ cells with (R)- or (S)-crizotinib, we found that the (S)-enantiomer did not exhibit any increased toxicity on non-transformed cells. Among a panel of human cancer cell lines, we consistently observed a strong antiproliferative effect for (S)-crizotinib, in line with its lower catalytic assay IC50 value. To explore the in vivo potential of (S)-crizotinib to impair tumour growth we performed mouse xenograft studies using SW480 cells. These experiments indicated that (S)-crizotinib, but not the (R)-enantiomer, was able to impair overall tumour progression as well as specifically reduce tumour volume by more than 50% (Fig. 4d, e, Extended Data Fig. 10a-c). This suggested that the two enantiomers have clearly diverse antitumoural profiles and was consistent with their distinct molecular mechanism of action.


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)

(S)-Crizotinib is a selective MTH1 inhibitor with in vivo anticancer activitya, The MTH1 inhibitors SCH51344 (5 μM) and (S)-crizotinib (2 μM), but not (R)-crizotinib (2 μM), induce DNA damage as indicated by an increase in 53BP1 foci and ATM autophosphorylation. Images are representative for three independent experiments (n = 3). b, Comet assay. Similar to MTH1 gene silencing both SCH51344 (5 μM) and (S)-crizotinib (2 μM), but not (R)-crizotinib (2 μM), induce DNA single strand breaks (MTM, mean tail moment). H2O2 was used as positive control (150 μM, 10 min). Images are representative for three independent experiments (n = 3), data are shown as mean ± SD. c, MTH1 overexpression reduces the number of DNA single strand breaks induced by SCH51344 and (S)-crizotinib. Compound concentrations are as in b. d, Results from SW480 mouse xenograft study. Effect on tumour growth following 35 days treatment with the MTH1 inhibitor (S)-crizotinib (25mg/kg q.d., s.c. daily, data are shown as mean ± SEM, n = 8/group). e, (S)-Crizotinib, but not (R)-crizotinib, impairs tumour growth in an SW480 colon carcinoma xenograft model (50mg/kg p.o., q.d.) Data show mean ± SEM, n=7-8 animals/group. Statistical analysis performed by 2-way repeat measurement ANOVA, Sidak’s multiple comparison; * p < 0.05 (S)-crizotinib vs control; † p < 0.05 (S)-crizotinib vs (R)-crizotinib. Images depict representative tumours for each treatment group (C, control). f, Proposed mechanism for MTH1-inhibitor-induced cancer cell death.
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Related In: Results  -  Collection

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Figure 4: (S)-Crizotinib is a selective MTH1 inhibitor with in vivo anticancer activitya, The MTH1 inhibitors SCH51344 (5 μM) and (S)-crizotinib (2 μM), but not (R)-crizotinib (2 μM), induce DNA damage as indicated by an increase in 53BP1 foci and ATM autophosphorylation. Images are representative for three independent experiments (n = 3). b, Comet assay. Similar to MTH1 gene silencing both SCH51344 (5 μM) and (S)-crizotinib (2 μM), but not (R)-crizotinib (2 μM), induce DNA single strand breaks (MTM, mean tail moment). H2O2 was used as positive control (150 μM, 10 min). Images are representative for three independent experiments (n = 3), data are shown as mean ± SD. c, MTH1 overexpression reduces the number of DNA single strand breaks induced by SCH51344 and (S)-crizotinib. Compound concentrations are as in b. d, Results from SW480 mouse xenograft study. Effect on tumour growth following 35 days treatment with the MTH1 inhibitor (S)-crizotinib (25mg/kg q.d., s.c. daily, data are shown as mean ± SEM, n = 8/group). e, (S)-Crizotinib, but not (R)-crizotinib, impairs tumour growth in an SW480 colon carcinoma xenograft model (50mg/kg p.o., q.d.) Data show mean ± SEM, n=7-8 animals/group. Statistical analysis performed by 2-way repeat measurement ANOVA, Sidak’s multiple comparison; * p < 0.05 (S)-crizotinib vs control; † p < 0.05 (S)-crizotinib vs (R)-crizotinib. Images depict representative tumours for each treatment group (C, control). f, Proposed mechanism for MTH1-inhibitor-induced cancer cell death.
Mentions: Since MTH1 is thought to prevent incorporation of oxidised nucleotides into DNA, we reasoned that our new MTH1 inhibitors should increase the content of genomic 8-oxoguanine, and thus induce DNA damage. Immunofluorescence staining for both 53BP1 and autophosphorylated ATM, specific markers for DNA damage, was increased in SW480 cells treated with MTH1 inhibitors (Fig. 4a, Extended Data Fig. 6a). 53BP1 foci, which we also observed in cells transfected with MTH1-siRNA, were enriched in nuclei of cells with higher levels of 8-oxoguanine due to increased genomic incorporation (Extended Data Fig. 6b, c). We also tried to quantify the oxidized nucleotides by HPLC-MS, however, due to high experimental background we failed to obtain reliable results. Since accumulation of 8-oxoguanine should activate base excision repair (BER)16 and induce DNA single strand breaks, we tested our inhibitors in an alkaline comet assay. Both (S)-crizotinib as well as SCH51344, but not (R)-crizotinib, yielded a significant tail moment, similar to cells transfected with MTH1-siRNA (Fig. 4b). Addition of the purified 8-oxoguanine- or 2-hydroxy-adenine-specific DNA glycosylases OGG1 and MUTYH, increased tail moments for (S)-crizotinib dramatically, providing evidence for strong accumulation of these lesions upon inhibitor treatment (Extended Data Fig. 6d). MTH1 overexpression significantly reduced the number of DNA single strand breaks induced by (S)-crizotinib as well as SCH51344, but not by H2O2 (Fig. 4c, Extended Data Fig. 6e) providing evidence for MTH1 being the functionally relevant target. To explore the role of p53 in the cellular response to MTH1 suppression16 we created a SW480 Tet-on system26 allowing for the inducible expression of p53 shRNAs and treated the cells with our inhibitors (Extended Data Fig. 7) which indicated a p53-independent mode of action. Treatment of SW480 cells expressing anti-MTH1 shRNA with the ATM- and ATR-inhibitors KU55933 and VE821, respectively, also did not display any differential effects (Extended Data Fig. 8). Similarly, ATM−/− MEFs were equally sensitive to MTH1 inhibitors as their ATM-proficient counterparts. Investigating cell lines bearing additional mutations in DNA repair genes we found that HCT116 deficient for p21 were particularly sensitive to (S)-crizotinib. We tested our inhibitors in BJ skin fibroblasts that were either wild type, immortalized by hTERT, or transformed by SV40T and/or mutant KRAS. Both SCH51344 and (S)-crizotinib exhibited highest toxicity toward the SV40T and KRAS-V12 cells (Extended Data Fig. 9). Importantly, when we treated wild type BJ cells with (R)- or (S)-crizotinib, we found that the (S)-enantiomer did not exhibit any increased toxicity on non-transformed cells. Among a panel of human cancer cell lines, we consistently observed a strong antiproliferative effect for (S)-crizotinib, in line with its lower catalytic assay IC50 value. To explore the in vivo potential of (S)-crizotinib to impair tumour growth we performed mouse xenograft studies using SW480 cells. These experiments indicated that (S)-crizotinib, but not the (R)-enantiomer, was able to impair overall tumour progression as well as specifically reduce tumour volume by more than 50% (Fig. 4d, e, Extended Data Fig. 10a-c). This suggested that the two enantiomers have clearly diverse antitumoural profiles and was consistent with their distinct molecular mechanism of action.

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