The E3 ligase Cbl-b and TAM receptors regulate cancer metastasis via natural killer cells

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Abstract

Tumour metastasis is the primary cause of mortality in cancer patients and remains the key challenge for cancer therapy1. New therapeutic approaches to block inhibitory pathways of the immune system have renewed hopes for the utility of such therapies2. Here we show that genetic deletion of the E3 ubiquitin ligase Cbl-b (casitas B-lineage lymphoma-b) or targeted inactivation of its E3 ligase activity licenses natural killer (NK) cells to spontaneously reject metastatic tumours. The TAM tyrosine kinase receptors Tyro3, Axl and Mer (also known as Mertk) were identified as ubiquitylation substrates for Cbl-b. Treatment of wild-type NK cells with a newly developed small molecule TAM kinase inhibitor conferred therapeutic potential, efficiently enhancing anti-metastatic NK cell activity in vivo. Oral or intraperitoneal administration using this TAM inhibitor markedly reduced murine mammary cancer and melanoma metastases dependent on NK cells. We further report that the anticoagulant warfarin exerts anti-metastatic activity in mice via Cbl-b/TAM receptors in NK cells, providing a molecular explanation for a 50-year-old puzzle in cancer biology3. This novel TAM/Cbl-b inhibitory pathway shows that it might be possible to develop a ‘pill’ that awakens the innate immune system to kill cancer metastases.

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Figure 1: Cbl-b mutant NK cells control metastatic melanomas.
Figure 2: Therapeutic anti-tumour activity of Cbl-b mutant NK cells.
Figure 3: TAM receptors are Cbl-b targets and characterization of a TAM receptor blocker.
Figure 4: Control of metastases by TAM receptor inhibition.

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Acknowledgements

We thank T. Hanada, R. Hanada, R. Karim and all other members of the Penninger laboratory for discussions and technical support. We thank all members of the IMP-IMBA BioOptics service facility for assistance in cell sorting and image quantification, and S. Soto and H. Popper for pathology analysis of tumour metastases. We thank A. L. Prieto, E. Vivier, D. Raulet and C. Melief for providing us with critical reagents and C. Martinez, A. Majoros and P. C. Esk for sharing reagents and for discussions. We also thank G. Kéri, L. Örfi and colleagues from Vichem Kft., Budapest, for their initial synthetic work on the quinoline-based Axl inhibitors, which served as the basis for the current rationale design of LDC1267. M.P. is supported by the European Research Council (ERC) and Era of Hope/DoD Innovator Award. J.M.P. is supported by grants from the Institute of Molecular Biotechnology (IMBA), the Austrian National Foundation, the Austrian Academy of Sciences, GEN-AU (AustroMouse), an Era of Hope/DoD Innovator Award and an EU ERC Advanced Grant.

Author information

All experiments, with the exceptions listed below, were performed by M.P. B.P. helped with mouse dissections, in vivo administration of LDC1267 and melanoma lung tumour quantifications. I.U. helped with 4T1 tumour experiments and performed Rae1 qPCR analysis on lung melanoma tumour samples. S.L. performed TC1 tumour experiments on Rag2−/− mice and the histological analysis of NeuT+ mammary gland tumours. A.C., C.S.-F., J.E., S.M., A.U., R.T., A.U. and B.M.K. developed the TAM small molecule inhibitor LDC1267 and characterized its kinase selectivity. S.W. and D.W. performed Cbl-b qRT–PCR, 51Cr-release NK cytotoxic assay, and perforin FACS analysis. R.N., S.J.C., T.G., R.H. and G.B. performed cell-free in vitro ubiquitylation experiments. W.Y.L. provided Cbl-b RING finger mutant mice. F.I., A.M.J. and J.P.F. provided reagents and expertise in key experiments. M.P. together with J.M.P. designed the experiments and wrote the manuscript.

Correspondence to Josef M. Penninger.

Ethics declarations

Competing interests

All co-authors from the Lead Discovery Center GmbH (Dortmund) and Max Planck Institute for Biochemistry (Martinsried) have filed a patent application covering the compound (LDC1267) used in our study; J.M.P. holds shares in a company that attempts to develop Cbl-b modulators.

Extended data figures and tables

Extended Data Figure 1 Cbl-b mutant NK cells are hyper-reactive.

a, Immunohistochemistry for NKp46 to detect NK cells in tumours isolated from Cbl-b+/+ and Cbl-b−/− mice at post-TC-1 inoculation day 14, when all experimental mice had similar tumour volumes (left). Tumour-infiltrating NK cells (%) in Cbl-b+/+ and Cbl-b−/− mice as detected by NKp46 immunohistochemistry (right). (mean ± s.e.m., n = 4 each). *P < 0.05 (Student’s t-test). b, Top, western blot for Cbl-b and Gapdh in FACS-sorted C373A+/+, C373A+/KI, and C373AKI/KI murine NK cells and the human NK cell line NK92. Bottom, quantitative Cbl-b messenger RNA expression in sorted primary wild-type murine and human NK and T cells. Cbl-b−/− T cells and H2O samples were used as negative controls. c, Ly49D-, Ly49F-, NKG2A/C/E- and NKG2D-expressing NK cells (%), as determined by FACS (mean ± s.e.m., n = 5–8). Gated on NK1.1+CD3ε or DX5+CD3ε NK cells. d, Percentage of splenic NK1.1+CD3ε cells in Cbl-b+/+, Cbl-b+/−, Cbl-b−/− and C373AKI/KI mice as determined by FACS. (mean ± s.e.m., n = 5–8). No significant differences were observed among any of the NK cell subpopulations analysed in c and d (one-way ANOVA). e, Percentage of IFN-γ-producing C373A+/KI and C373AKI/KI NK cells upon stimulation with anti-NKG2D antibodies (mean ± s.e.m., n = 3, representative of 4). **P < 0.01, ***P < 0.001 (Student’s t-test). f, g, IFN-γ-producing NK cells (%) upon NK1.1 stimulation of primary NK cells isolated from Cbl-b+/+ and Cbl-b−/− poly I:C-treated mice (f), and ex vivo-expanded (LAK) Cbl-b+/+ and Cbl-b−/− (f), as well as C373A+/KI and C373AKI/KI (g) NK cells (mean ± s.e.m., n = 3, representative of 3). *P < 0.05, **P < 0.01 (Student’s t-test). h, In vitro proliferation studies on wild-type ex vivo-expanded NK cells treated with anti-NK1.1 or anti-NKG2D antibodies as determined by cell tracer FACS analysis (left) and absolute cell number quantification (right). Interleukin (IL)-15 stimulation was used as positive control. i, j, In vitro proliferation of Cbl-b+/+ and Cbl-b−/− (i) and C373A+/KI and C373AKI/KI (j) NK cells treated with the indicated concentrations of plate-bound anti-NK1.1 and NKG2D antibodies (mean ± s.e.m., n = 3, representative of at least 3). *P < 0.05, **P < 0.01, ***P < 0.001 (Student’s t-test).

Extended Data Figure 2 Cbl-b controls NK cell cytotoxicity.

ac, In vitro NK cell cytotoxicity of C373A+/+ and C373AKI/KI (a) and Cbl-b+/+ and Cbl-b/ NK cells (b, c) towards YAC-1 targets as quantified by FACS analysis of TO-PRO-3 iodide+ YAC-1 cells (a, b) and 51Cr-release assay (c) (mean values ± s.e.m., n = 3). *P < 0.05, **P < 0.01, ***P < 0.001 (Student’s t-test). d, Percentages of IFN-γ-producing Cbl-b+/+ and Cbl-b/ NK cells upon incubation with YAC-1 targets (effector:target ratio 2:1, 6 h) (mean ± s.e.m., n = 3). *P < 0.05 (Student’s t-test). e, Degranulating murine NK cells (%) upon co-culture with YAC-1 targets, as determined by surface exposure of Lamp1 (mean ± s.e.m., n = 3). **P < 0.01 (Student’s t-test). f, Granzyme B release in the supernatant NK cells:YAC-1 co-cultures, at different effector:target ratios (mean ± s.e.m., n = 3). *P < 0.05 (Student’s t-test). g, Histogram for perforin expression in murine Cbl-b+/+ and Cbl-b/ NK cells upon incubation with YAC-1 cells (effector:target ratio 2:1). h, Left, cytotoxic activity of scrambled control siRNA- and CBL-B siRNA-treated human NKL cells towards Jurkat cells at different effector:target ratios 48 h after siRNA transfection. Right, western blot verifying CBL-B knockdown efficiency 8, 24 and 48 h after transfection is shown. **P < 0.01, ***P < 0.001 (Student’s t-test). For ag, ex vivo-expanded (LAK) NK cells were used. Data are representative of at least 2 independent experiments.

Extended Data Figure 3 Cbl-b, acting as an E3 ligase, controls NK-cell-mediated rejection of subcutaneous TC-1 tumours and melanomas.

a, Overall survival rates of untreated, NK1.1+-depleted, and NKG2D-blocked Cbl-b+/−, Cbl-b−/− and C373AKI/KI mice challenged with subcutaneous TC-1 tumours. Median survival values are shown (n = 5–14). *P < 0.05, **P < 0.01, ***P < 0.001 (log-rank test). No significant difference was found between the untreated and treated Cbl-b+/− groups. b, Kinetics of TC-1 tumour cell growth in individual control (top panels), NK1.1-depleted (middle panels) and NKG2D-blocked (bottom panels) Cbl-b+/+, Cbl-b−/− and C373AKI/KI mice. The numbers of experimental mice are indicated for each experiment and genotype (n). c, Histograms showing the expression of the NKG2D ligands Rae1 and Mult1 on TC-1 tumour cells (black). Isotype controls are shown in grey. d, Representative photographs showing B16F10 subcutaneous tumours in Cbl-b+/+ and Cbl-b−/− mice at day +16 post-tumour subcutaneous inoculation. e, Overall survival for Cbl-b+/+, Cbl-b−/− and C373AKI/KI mice subcutaneously challenged with 2.5 × 105 B16F10 melanoma cells (n = 5–7). **P < 0.01 Cbl-b−/− or C373AKI/KI versus Cbl-b+/+ mice (log-rank test). f, Representative FACS blots for NK1.1+ and CD3ε+ cell populations in C373A+/KI and C373AKI/KI littermate mice, confirming efficient splenic NK1.1+ cell depletion in mice receiving anti-NK1.1 antibodies. g, Representative images of untreated C373AKI/KI and NK1.1-depleted C373AKI/KI tumour-bearing mice at day +14 post-B16F10 inoculation. h, Overall survival of untreated and NK1.1-depleted Cbl-b+/− and Cbl-b−/− littermates subcutaneously challenged with 2.5 × 105 B16F10 cells (n = 6–8). *P < 0.05 Cbl-b−/− versus Cbl-b+/− and NK1.1-depleted Cbl-b−/− groups (log-rank test). i, Overall survival of untreated and NK1.1-depleted C373A+/KI and C373AKI/KI mice subcutaneously challenged with 2.5 × 105 B16F10 cells (n = 4–7). *P < 0.05 C373A+/KI versus C373AKI/KI, **P < 0.01 C373AKI/KI versus NK1.1-depleted C373AKI/KI (log-rank test).

Extended Data Figure 4 Cbl-b mutant NK cells can efficiently control melanoma lung metastases.

a, Automated quantification of lung and melanoma tumour areas using the Definiens Tissue software. An algorithm was developed to identify and categorize the lung images into normal lung tissue (yellow) and metastatic melanoma (green). The higher magnification inset in the right panel highlights the precision of the quantification. b, Tumour-to-lung ratios (%) in Cbl-b+/+(n = 14), Cbl-b+/− (n = 9), Cbl-b−/− (n = 11) and C373AKI/KI (n = 11) mice 21 days after intravenous injection of 2.5 × 105 B16F10 melanoma cells (mean ± s.e.m.). ***P < 0.001 Cbl-b+/+ versus Cbl-b−/− and C373AKI/KI groups (one-way ANOVA, Dunnett’s post-hoc test). c, Overall survival of Cbl-b+/+ (n = 13) , Cbl-b+/− (n = 5), Cbl-b−/− (n = 10) and C373AKI/KI (n = 10) mice intravenously challenged with 2.5 × 105 B16F10 cells. **P < 0.01 Cbl-b−/− versus Cbl-b+/+ groups. ***P < 0.001 C373AKI/KI versus Cbl-b+/+ groups (log-rank test). d, Representative lung photographs in Cbl-b+/+, Cbl-b+/−, Cbl-b−/− and C373AKI/KI mice 7 days after intravenous inoculation with 7.5 × 105 B16F10 cells. e, Quantification of total numbers of tumour foci in the lungs of Cbl-b+/+, Cbl-b+/−, Cbl-b−/− and C373AKI/KI mice treated as described in d (mean ± s.e.m., n = 5–6). *P < 0.05 Cbl-b−/− versus Cbl-b+/+, ***P < 0.001 C373AKI/KI versus Cbl-b+/+ mice (Student’s t-test). f, Histograms showing binding of the anti-NKG2D blocking antibodies to splenic NK cells in NKG2D-blocked Cbl-b−/− mice at the day of tumour inoculation (day 0). Anti-NKG2D antibodies were detected using anti-rat secondary antibodies. Histograms are gated on NK (NK1.1+CD3ε) and CD8+ T cells (CD3ε+CD8α+). Note that in these naive, unchallenged mice, the blocking antibodies do not bind to CD8+ T cells. Untreated Cbl-b−/− mice are shown as controls. g, Quantitative PCR showing Rae1 mRNA expression in B16F10 melanoma tumours isolated from the lung of tumour-bearing mice (day +16 after tumour challenge). Data are relative to the expression levels of Rae1 mRNA in healthy lung tumours isolated from non-tumour-bearing mice. h, Photographs of individual lungs of Cbl-b+/+, Cbl-b+/−, Cbl-b−/− and C373AKI/KI mice treated with the anti-NKG2D blocking antibodies and intravenously challenged with B16F10. Images are from day +21. Photographs for untreated mice are shown in Fig. 1e. i, Tumour-to-lung ratios (%) of control and anti-NKG2D-blocked Cbl-b+/+, Cbl-b+/−, Cbl-b−/− and C373AKI/KI mice 21 days after B16F10 tumour inoculation (mean ± s.e.m., n = 5–12). *P < 0.05 Cbl-b−/− versus Cbl-b−/− NKG2D-blocked mice, **P < 0.01 C373AKI/KI versus NKG2D-blocked C373AKI/KI mice (Student’s t-test).

Extended Data Figure 5 The role CD8+ cells and perforin-mediated cytotoxicity in controlling melanoma metastasis.

a, Photographs of melanoma metastases found in the pancreas, kidney, testis and lymph node of Cbl-b+/+ mice 21 days post-tumour inoculation. Similar results were observed in Cbl-b+/− mice. b, Prevalence of extrapulmonary melanoma metastases in Cbl-b+/+, Cbl-b+/−, Cbl-b−/− and C373AKIKI mice, defined by the presence of macroscopically visible melanoma tumours in any organ other than the lung, regardless of the number of organs affected per mouse. Data are from day +21 post-tumour challenge. *P < 0.05 Cbl-b−/− and C373AKIKI versus Cbl-b+/+ groups (Chi-squared test). c, Numbers of extrapulmonary metastases per mouse in Cbl-b+/+, Cbl-b+/−, Cbl-b−/− and C373AKI/KI mice (day +21). Data from individual mice (n = 12–18) and median values are shown (horizontal lines). *P < 0.05 Cbl-b−/− or C373AKI/KI versus Cbl-b+/+ group (Mann–Whitney U-test). d, Presence of melanoma metastases (%) in different tissues of Cbl-b+/+ (n = 16), Cbl-b+/− (n = 10), Cbl-b−/− (n = 9) and C373AKI/KI (n = 11) mice (day +21). e, Representative lung photographs of untreated and NK1.1+-cell-depleted Cbl-b+/− and Cbl-b−/− mice 28 days after intravenous injection of a 10-times lower dose of B16F10 tumour cells (2.5 × 104). f, Tumour-to-lung ratios (%) of non-depleted and NK1.1+-depleted Cbl-b+/− and Cbl-b−/− mice treated as described in e. Data are mean values ± s.e.m. at the experimental end point. (n = 3–4). ***P < 0.001, NS, not significant (Student’s t-test). g, Number of extrapulmonary tumour metastases in control or NK1.1-depleted Cbl-b+/− and Cbl-b−/− mice 28 days after injection of 2.5 × 104 B16F10 tumour cells. Horizontal lines are median values. h, Representative lung photographs of CD8+-cell-depleted Cbl-b+/+, Cbl-b+/−, Cbl-b−/− and C373AKI/KI mice 21 days after intravenous injection of B16F10 tumour cells. i, Tumour-to-lung ratios (%) of non-depleted Cbl-b+/+ (n = 12), Cbl-b+/− (n = 6), Cbl-b−/− (n = 9) and C373AKI/KI (n = 7) and CD8+-depleted Cbl-b+/+(n = 6), Cbl-b+/− (n = 4), Cbl-b−/− (n = 4) and C373AKI/KI (n = 4) mice. Data are mean values ± s.e.m. at the experimental end point. Of note, we observed a reduction in the tumour-to-lung ratios of Cbl-b+/+ and Cbl-b+/− mice following in vivo depletion of CD8+ cells, a finding that requires further explorations. j, Numbers of extrapulmonary melanoma metastases in Cbl-b+/− Prf1+/+ (n = 10), Cbl-b−/− Prf1+/+ (n = 7), Cbl-b+/− Prf1−/− (n = 7) and Cbl-b−/− Prf1−/− (n = 11) mice at the experimental end point (day +21). *P < 0.05, ***P < 0.01 (Mann–Whitney U-test).

Extended Data Figure 6 Mice deficient in Cbl-b can control the growth of NeuT+ mammary and metastatic lung tumours.

a, Kinetics of NeuT+ mammary tumour growth in Cbl-b+/− NeuT+ and Cbl-b−/− NeuT+ mice (n = 27 for each). Data are mean values ± s.e.m. *P < 0.05, **P < 0.01 (Student’s t-test). b, Onset of palpable mammary tumours in Cbl-b+/− NeuT+ (n = 30) and Cbl-b−/− NeuT+ (n = 43) transgenic mice. The median tumour onset for Cbl-b+/− NeuT+ and Cbl-b−/− NeuT+ mice was 107 and 110 days after birth, respectively. P > 0.05, not significant (log-rank test). c, Overall survival for Cbl-b+/− NeuT+ and Cbl-b−/− NeuT+ mice (n = 27 for each). Mice were euthanized when total tumour volume of all affected mammary glands reached 1,500 mm3. **P < 0.01 (log-rank test). d, Histological appearance of isolated mammary glands in 18-, 20-, 21-, 22- and 24-week-old Cbl-b+/− NeuT+ and Cbl-b−/− NeuT+ littermates. Red arrows indicate mammary tumours, black arrows the axillary lymph nodes. Haematoxylin and eosin staining. Scale bars, 1 mm. e, Representative images for the automated quantification of metastatic lung mammary carcinomas in mice bearing NeuT+ mammary tumours using a customized algorithm in Definiens Tissue Software to identify and categorize two areas: normal lung tissue (blue) and metastatic mammary carcinoma (green). f, g, Numbers of tumour foci (f) and tumour-to-lung area (%; g) in lungs of Cbl-b+/− NeuT+ and Cbl-b−/− NeuT+ mice bearing NeuT+ mammary tumours (n = 17/20). At least two lung images were quantified per mouse (≥350 μm apart). *P < 0.05 (Mann–Whitney U-test, lines are median values). h, Quantification of NK cells infiltrating the metastatic mammary lung carcinomas of Cbl-b+/− NeuT+ and Cbl-b−/− NeuT+ mice, as detected by immunohistochemistry for NKp46 and quantified using the Definiens Software (n = 13–15 per genotype, lines are median). *P < 0.05 (Mann–Whitney U-test). i, Images showing specific staining of NK cells in spleen using the anti-NKp46 antibody. Untreated and NK-cell-depleted animals were used as positive and negative controls, respectively.

Extended Data Figure 7 Cytotoxic NK cells are required for the efficient control of mammary cancer metastases to the lung.

a, Flow cytometry blots for DX5+ and CD3ε+ cell populations in untreated and anti-asialo GM1-treated Cbl-b−/− NeuT+ mice confirming efficient splenic NK cell depletion at the experimental end point. Numbers indicate percentages of DX5+ NK cells in the respective gates. b, Tumour-to-lung area (%) for metastatic mammary carcinoma in the lungs of control and anti-asialo GM1-treated Cbl-b−/− NeuT+ mice (n = 11/10). Median values are shown with a horizontal line. *P < 0.05 (Mann–Whitney U-test). c, Representative H&E-stained lung sections of control and anti-asialo GM1-treated Cbl-b+/− NeuT+ mice showing metastatic mammary carcinomas (arrows). d, e, Relative tumour-to-lung areas (%) (d) and numbers of metastatic tumour foci (e) in the lungs of control and anti-asialo GM1-treated Cbl-b+/− NeuT+ mice (n = 6–7, two sections per mouse were quantified). *P < 0.05, **P < 0.01 (Mann–Whitney U-test). Lines are median values. f, g, Numbers of tumour foci (f) and relative tumour-to-lung area (%) (g) in the lungs of Cbl-b−/− Prf1+/+NeuT+, Cbl-b−/− Prf1+/− NeuT+ and Cbl-b−/− Prf1−/− NeuT+ mice (n = 5–6, lines are median values. At least two sections per mouse were quantified. *P < 0.05 (Mann–Whitney U-test). h, Representative H&E-stained lung sections of Cbl-b−/− Prf1+/+NeuT+, Cbl-b−/− Prf1+/− NeuT+ and Cbl-b−/− Prf1−/− NeuT+ mice showing metastatic mammary carcinomas (arrows). Note the increase in metastatic tumour foci and total tumour area even in the absence of one copy of the perforin gene.

Extended Data Figure 8 TAM tyrosine kinase receptors are novel ubiquitylation targets of Cbl-b.

a, Out of 9,000 human proteins tested, TYRO3 had the highest CBL-B-mediated ubiquitylation signal. Signal intensities are shown for the corresponding TYRO3 spots in the protein arrays incubated with the E2 enzyme without CBL-B, the CBL-B(C373A) mutant and wild-type CBL-B proteins (in duplicates). Data are shown as mean values. RFU, relative fluorescent units. b, In vitro ubiquitylation of recombinant Flag-tagged TYRO3, AXL and MER in the presence (left) and absence (right) of CBL-B. Blots were probed with anti-Flag antibodies. c, Immunoprecipitation showing time-dependent recruitment of CBL-B to TAM tyrosine kinase receptors in HeLa cells upon stimulation with His-tagged GAS6 (top panel). Input levels of CBL-B and β-actin are shown as controls. d, GAS6-induced AXL ubiquitylation depends on CBL-B expression. HeLa cells were transfected with scrambled siControl or siCBL-B and then stimulated with 450 ng ml−1 GAS6 for 15 min or left untreated. Lysates were immunoprecipitated with anti-Axl antibodies. Blots were then probed using anti-ubiquitin (Ub) and anti-Axl antibodies. The location of the mature form of AXL (140 kDa) is shown with an arrow. AXL, CBL-B and tubulin protein levels are shown to control for the input (bottom panels). e, Representative histograms showing equal expression of Tyro3, Axl and Mer receptors at the cell surface of freshly isolated NK1.1+CD3ε splenic Cbl-b+/+ Cbl-b−/− and C373AKI/KI NK cells. Background staining with control isotype antibodies is shown for each blot in grey. f, In vitro proliferation of Cbl-b+/− and Cbl-b−/− NK cells stimulated with anti-NKG2D antibodies (30 μg ml1) in the presence of different concentrations of Gas6 (mean values ± s.e.m., n = 4). *P < 0.05 compared to the reference (no Gas6) value in the Cbl-b+/− group. No significant difference was detected among the Cbl-b−/− NK cells treated with different concentrations of Gas6 (one-way ANOVA, Dunnett’s post-hoc test). g, Representative histograms showing equal expression of AXL at the cell surface of HeLa cells that were transfected with scrambled siControl or siCBL-B. Data were collected 48 h after siRNA transfection. h, AXL surface expression in siControl or siCBL-B HeLa cells stimulated with 200 ng ml1 recombinant human GAS6 for the indicated time periods. Western blots show efficient downregulation of CBL-B protein levels in HeLa cells 48 h after siRNA transfection (right panel). *P < 0.05 (Student’s t-test).

Extended Data Figure 9 Biochemical and cellular selectivity of LDC1267.

a, Target proteins of LDC1267 in Hs578T cells. Determined Kd values are plotted on a logarithmic scale. All identified targets were protein kinases and are ranked from low (top) to high (bottom) Kd values (in μM). The experimental procedure is summarized at http://www.evotec.com/uploads/media_library/31/2013-09_S6_Proteomics.pdf. b, c, Exemplary dose–response curve of LDC1267 inhibition of ectopically expressed Tyro3 phosphorylation. c, Quantification of the western blot depicted in b. After transfection of HEK293 cells with Tyro3 expression plasmids cells were incubated with LDC1267 or DMSO for 1 h. Protein lysates were separated by SDS–PAGE and blotted to a PVDF membrane. Tyro3 levels as well as its phosphorylation state were determined by Red/Alexa Fluor 680-labelled (Tyro3 expression) and green/IR800-labelled antibodies (tyrosine phosphorylation). d, IC50 values (y axis in μM) of LDC1267 on a panel of 93 cancer cell lines and two primary cells (x axis, IMR90 and human peripheral blood mononuclear cells) in a proliferation assay. After incubation for 72 h with LDC1267, CellTiterGlow reagent (Promega) was used to determine the proliferation relative to the corresponding DMSO control. e, Histogram confirming the high expression of the NKG2D ligand Rae1 at the cell surface of the genetically engineered RMA-Rae1 cell line. Staining with an isotype control antibody and basal expression of Rae1 in RMA cells are also shown. RMA and RMA-Rae1 cells lines were used for studying NKG2D-dependent NK cell cytotoxic responses in vivo. f, Protocol used to test the effects of LCD1267 in an NK cell adoptive transfer model for the treatment of metastatic melanoma. Wild-type C57BL/6J (B6) recipient mice were injected intravenously with 2.5 × 105 B16F10 melanoma cells at day 0. At day +1 and +4, recipient mice received 1.0 × 105 sorted syngeneic Cbl-b-sufficient or Cbl-b−/− NK cells that were either pre-treated for 2.5 h ex vivo with vehicle (DMSO) or LDC1267 (2.5 μM). A control untreated group received tumour cells but did not receive NK cells (untreated B6). Mice were euthanized at day +14 post-tumour inoculation and lung tumour foci and total tumour areas in lungs were quantified.

Extended Data Figure 10 Oral LDC1267 and warfarin administration reduces tumour metastases.

a, Kinetics of primary 4T1 tumour cell growth in the mammary fat pad of control and anti-asialo GM1-treated mice that received LDC1267 or vehicle (mean ± s.e.m., n = 6–9 mice per group). NS, not significant (one-way ANOVA). b, Representative images for the quantification of 4T1 liver micro-metastases using the Definiens Tissue Software to identify and categorize normal lung tissue (blue) and metastatic mammary carcinoma (green, arrows). c, Plasma concentrations of LDC1267 in mice bearing 4T1 metastatic tumours and treated daily by intraperitoneal injections with vehicle or LDC1267 (20 mg kg−1). Data are from 12 h after the last treatment (day +16). Horizontal line indicates median value. ND, not detectable. d, Plasma concentrations of LDC1267 in mice bearing 4T1 metastatic tumours treated daily by oral gavage with vehicle or LDC1267 (100 mg kg−1). Data are from 11 h after the last treatment (day +21). Horizontal line indicates median value. e, f, Relative sizes (e) and numbers (f) of 4T1 liver micro-metastases in syngeneic mice treated with vehicle or LDC1267 (100 mg kg−1) via oral gavage. Mean values ± s.e.m. are shown on day +21 after initiation of LDC1267 therapy (day +27 after orthotopic tumour inoculation into the mammary fat pad). ***P < 0.001 (Student’s t-test, n = 10 mice each). g, Representative photographs of 4T1 liver micro-metastases in mice treated as described in ef. Arrows indicate micro-metastases. h, Prothrombine times (mean ± s.d.) in warfarin (0.5 mg l−1)- and vehicle-treated mice, showing that the low dose of warfarin used does not cause a coagulopathy, confirming previous results21,22. Of note, we did not observe either spontaneous or excessive bleeding in warfarin-treated mice as compared to vehicle controls. Data are from mice treated orally (in drinking water) for 21 days. i, Numbers of extrapulmonary metastases in individual vehicle-treated Cbl-b+/+ (n = 23), Cbl-b+/− (n = 11), Cbl-b−/− (n = 7) and C373AKI/KI (n = 12) and warfarin-treated Cbl-b+/+ (n = 19), Cbl-b+/− (n = 11), Cbl-b−/− (n = 9) and C373AKI/KI (n = 11) mice at day +16 post B16F10 intravenous inoculation. *P < 0.05 comparing the control and warfarin-treated groups in either Cbl-b+/+ or Cbl-b+/− mice (Mann–Whitney U-test). NS, not significant. Horizontal lines represent median values. j, Representative photographs of the individual lungs of NK1.1-sufficient and NK1.1-depleted vehicle-treated and warfarin-treated Cbl-b+/+ mice bearing lung metastatic melanomas. Data are from day +16 after B16F10 intravenous inoculation. k, In vivo NK cell cytotoxicity in vehicle- and warfarin-treated Cbl-b+/+ mice shown as relative percentages of RMA-Rae1 and RMA-S cells recovered from the peritoneal cavity 24 h after intraperitoneal injection of a 1:1 ratio with the NK-resistant cell line RMA (mean ± s.e.m., n = 5 each). Mice were treated with vehicle and warfarin for 7 days before inoculation of RMA, RMA-S and RMA-Rae1 cells. *P < 0.05 (Student’s t-test).

Supplementary information

Supplementary Table 1

This file contains Supplementary Table 1, LDC1267 kinase selectivity. This table shows remaining relative activity (compared to DMSO control) in a selectivity panel of 456 kinases treated with 1µM LDC1267 as determined by DiscoverX's KINOMEscan technology. The kinase identities and their corresponding gene symbols are shown. The red-green color gradient correlates with the degree of inhibition: from red (1% remaining activity) to green (100% remaining activity). (PDF 244 kb)

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Paolino, M., Choidas, A., Wallner, S. et al. The E3 ligase Cbl-b and TAM receptors regulate cancer metastasis via natural killer cells. Nature 507, 508–512 (2014) doi:10.1038/nature12998

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