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

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.

Extended Data Figure 1. Confirmation of MTH1 as the main cellular target of SCH51344

a, 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).

Extended Data Figure 2. (S)-Crizotinib target specificity

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, K_d binding constants of both crizotinib enantiomers for the (R)-crizotinib cognate targets ALK, MET, and ROS1. Data are shown as mean ± SEM (n = 2). c, Pharmacologic c-MET kinase inhibition by a highly potent inhibitor (JNJ-38877605, 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 toward (S)-crizotinib. Data are shown as mean ± SEM 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/condition), and each replicate was analysed in two technical replicates.

Extended Data Figure 3. KINOMEscan results for both crizotinib enantiomers

Screening of both (R)- and (S)-crizotinib against a panel of 456 recombinant human protein kinases indicates a dramatic difference in the ability of the two enantiomers to bind kinases. (R)-crizotinib has high affinity toward 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).

Extended Data Figure 4. Co-crystal structures of (S)- and (R)-crizotinib bound to MTH1

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 2F_o-F_c 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.

Extended Data Figure 5. Data collection and refinement statistics

a, Crystallization of MTH1 complexes. b, Data collection and refinement statistics.

Extended Data Figure 6. MTH1 suppression by siRNA or small molecule inhibitors induces DNA damage

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 ± SD (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 knock-down of MTH1 also induces formation of 53BP1 foci in SW480 cells. Images are representative and data are shown as mean ± SD based on three independent experiments (n = 3) (P < 0.05, t-test). c, Formation of 53BP1 foci correlates with increased 8-oxoguanine 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-oxoguanine- 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 ± SEM 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 as compared to empty vector transfected cells. Concentrations used are as in c. Numbers depict MTM ± SD, statistical significance was determined using the Holm-Sidak method (p < 0.05) (n = 2).

Extended Data Figure 7. MTH1 inhibitor efficacy is not affected by loss of p53

a, Western blot evaluation of p53-shRNA knock-down 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 two days either with or without doxycycline, plated in triplicate in 6-well plates, and drugs added 24 h later. Colonies were stained with crystal violet and quantified using UV absorbance after dye solubilisation with ethanol. Data are shown as mean ± SEM and are based on three independent experiments (n = 3).

Extended Data Figure 8. Interplay of MTH1 activity and DNA damage proteins

a, Stable knock-down of MTH1 does not alter SW480 sensitivity toward ATM (KU55933) or ATR (VE821) kinase inhibition. Data are shown as mean ± SEM and are based on three independent experiments (n = 3). b, Conversely, ATM status does not affect MTH1 inhibitor efficacy in immortalised MEFs. Data are shown as mean ± SEM 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 toward MTH1 inhibitors, however, p21^−/− cells are more sensitive, in particular to the more potent MTH1 inhibitor (S)-crizotinib (top). Data are shown as mean ± SEM and are based on three independent experiments (n = 3). Similarly, BRCA2 function does not alter MTH1 inhibitor sensitivity of VC-8 cells (bottom). Data are shown as mean ± SEM and are based on three independent experiments (n = 3).

Extended Data Figure 9. MTH1 inhibitors exert selective toxicity toward transformed cells

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 ± SEM 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.

Extended Data Figure 10. Xenograft supplementary data and Oncomine MTH1 meta-analysis

a, Mouse haematology and liver/heart/kidney parameters comparing treatment versus controls. SCID mice (n = 8/group) were subcutaneously administered vehicle or (S)-crizotinib (25mg/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 (50mg/kg p.o., q.d.), (S)-crizotinib (50mg/kg p.o., q.d.) or vehicle on tumour volume at day 26 in SW480 xenograft mice. Individual data is shown, n = 7-8 animals/group. Statistical analysis performed by 2-way repeat measurement ANOVA, followed by Sidak’s multiple comparison. c, Effect of treatment on body weight. Data show mean ± SEM. 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.

Figure 1. MTH1 is the target of SCH51344

a, Representation of the chemical proteomic workflow. b, Sructures of SCH51344 (1) and the probe used for affinity purification (2). c, Results from MS-based proteomic affinity purification experiment using SAINT and competition analysis. Data shown are based on two independent experiments for each condition (n = 2/condition), and each replicate was analysed in two technical replicates. d, ITC data for MTH1 with SCH51344. The measured K_d was 49 nM (n = 1). e, SCH51344 inhibits hydrolysis of the MTH1 substrates dGTP, 8-oxo-dGTP, and 2-OH-dATP, respectively. Data are shown for two technical replicates ± SEM and representative for at least duplicate experiments (n ≥ 2). f, Silencing of MTH1 by siRNA impairs colony formation of KRAS-positive SW480 (top) and DLD1 (bottom) cells. Data shown as mean ± SEM and images are representative for triplicate experiments (n = 3) (P < 0.05, t-test). Asterisk denotes unspecific band. g, MTH1 overexpression as monitored by real-time PCR (left) restores SW480 cell viability upon SCH51344 treatment (right). Data shown as mean ± SEM and images are representative for three independent experiments (n = 3).

Figure 2. (S)-Crizotinib is a nanomolar MTH1 inhibitor

a, MTH1 catalytic assay. Data are shown for both crizotinib enantiomers and the racemic mixture at 100 μM dGTP. Results indicate two technical replicates ± SEM representative for at least duplicate experiments (n ≥ 2). b, ITC for MTH1 with (R)- and (S)-crizotinib (n = 1). c, (S)-Crizotinib inhibits colony formation of PANC1 and SW480 cells. Images are representative for three independent experiments (n = 3). d, Comparison of antiproliferative efficacy of (S)-crizotinib versus SCH51344 against SW480 cells. Data shown as mean ± SEM for three independent experiments (n = 3). e, Cellular thermal shift assay MTH1 target engagement by (S)-crizotinib in intact KRASV12-expressing BJ cells. Images are representative of two independent experiments (n = 2). f, The (S)-crizotinibaffinity probe selectively binds MTH1, but not ALK, in SW480 lysates, whereas the (R)-enantiomer exerts inverse properties.

Figure 3. Specificity and MTH1 co-crystal structure of (S)-crizotinib

a, Comparison of (S)-crizotinib specificity versus SCH51344 and b, (R)-crizotinib. MTH1 is the only shared target with SCH51344 and is specific to (S)-crizotinib when compared to (R)-crizotinib. Data represent two independent experiments for each condition (n = 2/condition), and each replicate was analysed in two technical replicates. c, Co-crystal structure of (S)-crizotinib and MTH1. MTH1 is in pink with light green alpha-helices and the loops covering the binding site in blue. Hydrogen-bonding interactions are shown by dashed red lines. d, MTH1 interactions with (R)- and (S)-crizotinib. Left panel shows (R)-crizotinib in yellow; the thinner lines indicate part of the (R)-crizotinib that was not resolved in the electron density. Right panel shows (S)-crizotinib in cyan; alternate protein conformations in the absence of (S)-crizotinib are shown in dark green.

Figure 4. (S)-Crizotinib is a selective MTH1 inhibitor with in vivo anticancer activity

a, 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.