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

Abstract

Tumour metastasis is the primary cause of mortality in cancer patients and remains the key challenge for cancer therapy. New therapeutic approaches to block inhibitory pathways of the immune system have renewed hopes for the utility of such therapies. 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 biology. 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.

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

a, Immunohistochemistry for NKp46 to detect NK cells in tumors isolated from Cbl-b^+/+ and Cbl-b^–/– mice at post TC-1 inoculation day 14, when all experimental mice had similar tumor volumes (left). Tumor 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, Western blot for Cbl-b and GAPDH in FACS sorted C373A^+/+, C373A^+/KI, and C373A^KI/KI murine NK cells and the human NK cell line NK92 (upper panel). Quantitative Cbl-b mRNA expression in sorted primary wild-type murine and human NK and T cells (bottom panels). Cbl-b^-/- T cells and H₂0 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 C373A^KI/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 analyzed in c and d (One-way ANOVA). e, Percentage of IFN-γ producing C373A^+/KI and C373A^KI/KI NK cells upon stimulation with anti-NKG2D Abs. (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 C373A^KI/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). IL-15 stimulation was used as positive control. i-j, In vitro proliferation of Cbl-b^+/+ and Cbl-b^-/- (i), and C373A^+/KI and C373A^KI/KI (j) NK cells treated with the indicated concentrations of plate-bounded 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.

a-c, In vitro NK cell cytotoxicity of C373A^+/+ and C373A^KI/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, and ***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 hours). (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 LAMP-1 (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, Cytotoxic activity of scrambled control-siRNA and Cbl-b siRNA treated human NKL cells towards Jurkat cells at different effector:target ratios 48 hours after siRNA transfection. Western blot verifying Cbl-b knockdown efficiency 8, 24, and 48 hours after transfection is shown (left panel). **P<0.01, ***P<0.001 (Student’s t-test). For a-g ex vivo expanded (LAK) NK cells were used. Data is 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 tumors and melanomas.

a, Overall survival rates of untreated, NK1.1⁺-depleted, and NKG2D-blocked Cbl-b^+/-, Cbl-b^-/- and C373A^KI/KI mice challenged with subcutaneous TC-1 tumors. 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 tumor cell growth in individual control (upper panels), NK1.1-depleted (middle panels) and NKG2D-blocked (bottom panels) Cbl-b^+/+, Cbl-b^-/-, and C373A^KI/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 tumor cells (black). Isotype controls are shown in grey. d, Representative photographs showing B16F10 subcutaneous tumors in Cbl-b^+/+ and Cbl-b^-/- mice at day +16 post-tumor s.c. inoculation. e, Overall survival for Cbl-b^+/+, Cbl-b^-/-, and C373A^KI/KI mice subcutaneously challenged with 2.5x10⁵ B16F10 melanoma cells (n=5-7). **P<0.001 Cbl-b^-/- or C373A^KI/KI vs. Cbl-b^+/+ mice (log-rank test). f, Representative FACS blots for NK1.1⁺ and CD3ε⁺ cell populations in C373A^+/KI and C373A^KI/KI littermate mice, confirming efficient splenic NK1.1⁺ cell depletion in mice receiving anti-NK1.1 antibodies. g, Representative images of untreated C373A^KI/KI and NK1.1-depleted C373A^KI/KI tumor-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 2.5x10⁵ B16F10 cells (n=6-8). *P<0.05 Cbl-b^-/- vs Cbl-b^+/- and NK1.1-depleted Cbl-b^-/- groups (log-rank test). i, Overall survival of untreated and NK1.1-depleted C373A^+/KI and C373A^KI/KI mice subcutaneously challenged with 2.5x10⁵ B16F10 cells (n=4-7). *P<0.05 C373A^+/KI vs C373A^KI/KI, **P<0.01 C373A^KI/KI vs NK1.1-depleted C373A^KI/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 tumor 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 lower panel highlights the precision of the quantification. b, Tumor-to-lung ratios (%) in Cbl-b^+/+(n=14), Cbl-b^+/- (n=9), Cbl-b^-/- (n=11) and C373A^KI/KI (n=11) mice 21 days after intravenous injection of 2.5x10⁵B16F10 melanoma cells (mean ± s.e.m.). ***P<0.001 Cbl-b^+/+ vs Cbl-b^-/- and C373A^KI/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 C373A^KI/KI (n=10) mice i.v. challenged with 2.5x10⁵ B16F10 cells. **P<0.01 Cbl-b^-/- vs. Cbl-b^+/+ groups. ***P<0.001 C373A^KI/KI vs. Cbl-b^+/+ groups (log-rank test). d, Representative lung photographs in Cbl-b^+/+, Cbl-b^+/-, Cbl-b^-/-, and C373A^KI/KI mice 7 days after i.v. inoculation with 7.5x10⁵ B16F10 cells. e, Quantification of total numbers of tumor foci in the lungs of Cbl-b^+/+, Cbl-b^+/-, Cbl-b^-/-, and C373A^KI/KI mice treated as described in d (mean ± s.e.m., n=5-6). *P<0.05 Cbl-b^-/- vs Cbl-b^+/+ and ***P<0.001 C373A^KI/KI vs. 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 tumor inoculation (day 0). Anti-NKG2D antibodies were detected using anti-rat secondary Abs. Histograms are gated on NK (NK1.1⁺CD3ε^-) and CD8⁺ T cells (CD3ε⁺CD8α⁺). Note that in this naïve 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 tumors isolated from the lung of tumor bearing mice (day +16 after tumor challenge). Data is relative to the expression levels of Rae1 mRNA in healthy lung tumors isolated from non-tumor bearing mice. h, Photographs of individual lungs of Cbl-b^+/+, Cbl-b^+/-, Cbl-b^-/-, and C373A^KI/KI mice treated with the anti-NKG2D blocking Abs and i.v. challenged with B16F10. Images are from day +21. Photographs for untreated mice are shown in Fig. 1e. i, Tumor-to-lung ratios (%) of control and anti-NKG2D-blocked Cbl-b^+/+, Cbl-b^+/-, Cbl-b^-/-, and C373A^KI/KI mice 21 days after B16F10 tumor-inoculation (mean ± s.e.m., n=5-12). *P<0.05 Cbl-b^-/- vs. Cbl-b^-/- NKG2D-blocked mice; **P<0.01 C373A^KI/KI vs. NKG2D-blocked C373A^KI/KI mice (Student’s t-test).

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

a, Photographs of melanoma metastases found in the pancreas, kidney, testis, and lymph node of Cbl-b^+/+ mice 21 days post-tumor inoculation. Similar results were observed in Cbl-b^+/- mice. b, Prevalence of extrapulmonary melanoma metastases in Cbl-b^+/+, Cbl-b^+/-, Cbl-b^-/-, and C373A^KIKI mice, defined by the presence of macroscopically visible melanoma tumors in any organ other than the lung, regardless of the number of organs affected per mouse. Data are from day +21 post-tumor challenge. *P<0.05 Cbl-b^-/- and C373A^KIKI vs. Cbl-b^+/+ groups (Chi-square test). c, Numbers of extra-pulmonary metastases per mouse in Cbl-b^+/+, Cbl-b^+/-, Cbl-b^-/-, and C373A^KI/KI mice (day +21). Data from individual mice (n=12-18) and median values are shown (horizontal lines). *P<0.05 Cbl-b^-/- or C373A^KI/KI vs. Cbl-b^+/+ group (Mann-Whitney test). d, Presence of melanoma metastases (%) in different tissues of Cbl-b^+/+ (n=16), Cbl-b^+/- (n=10), Cbl-b^-/- (n=9), and C373A^KI/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 i.v. injection of a 10 times lower dose of B16F10 tumor cells (2.5x10⁴). f, Tumor-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 endpoint. (n=3-4). ***P<0.001, n.s.=not significant (Student’s t-test). g, Number of extrapulmonary tumor metastases in control or NK1.1-depleted Cbl-b^+/- and Cbl-b^-/- mice 28 days after injection of 2.5x10⁴ B16F10 tumor cells. Horizontal lines are median values. h, Representative lung photographs of CD8⁺ cell-depleted Cbl-b^+/+, Cbl-b^+/-, Cbl-b^-/- and C373A^KI/KI mice 21 days after i.v. injection of B16F10 tumor cells. i, Tumor-to-lung ratios (%) of non-depleted Cbl-b^+/+ (n=12), Cbl-b^+/- (n=6), Cbl-b^-/- (n=9), and C373A^KI/KI (n=7) and CD8⁺-depleted Cbl-b^+/+(n=6), Cbl-b^+/- (n=4), Cbl-b^-/- (n=4), and C373A^KI/KI (n=4) mice. Data are mean values ± s.e.m. at the experimental endpoint. Of note, we observed a reduction in the tumor/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 endpoint (day +21). *P<0.05, **P<0.01 (Mann-Whitney test).

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

a, Kinetics of NeuT⁺ mammary tumor 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 tumors in Cbl-b^+/-NeuT⁺ (n=30) and Cbl-b^-/-NeuT⁺ (n=43) transgenic mice. The median tumor 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 tumor volume of all affected mammary glands reached 1500mm³. **P<0.01 (log-rank test). d, Histological appearance of isolated mammary glands in 18, 20, 21, 22, and 24 weeks old Cbl-b^+/-NeuT⁺ and Cbl-b^-/-NeuT⁺ littermates. Red arrows indicate mammary tumors, black arrows the axillary lymph nodes. H&E staining. Bars, 1mm. e, Representative images for the automated quantification of metastatic lung mammary carcinomas in mice bearing NeuT⁺ mammary tumors using a customized algorithm in Definiens Tissue Software to identify and categorize 2 areas: normal lung tissue (blue), and metastatic mammary carcinoma (green). f,g, Numbers of tumor foci (f) and tumor-to-lung area (%) (g) in lungs of Cbl-b^+/-NeuT⁺ and Cbl-b^-/-NeuT⁺ mice bearing NeuT⁺ mammary tumors (n=17/20). At least 2 lung images were quantified per mouse (≥ 350μm apart). *P<0.05 (Mann-Whitney 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, line are median). *P<0.001 (Mann-Whitney 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 endpoint. Numbers indicate percentages of DX5⁺ NK cells in the respective gates. b, Tumor-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 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 tumor-to-lung areas (%) (d) and numbers of metastatic tumor foci (e) in the lungs of control and anti-asialo GM1-treated Cbl-b^+/-NeuT⁺ mice (n=6-7, 2 sections per mouse were quantified). *P<0.05 **P<0.01 (Mann-Whitney test). Lines are median values. f-g, Numbers of tumor foci (f) and relative tumor-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 2 sections per mouse were quantified. *P<0.05 (Mann-Whitney 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 tumor foci and total tumor 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 9000 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 panel) and absence (right panel) of Cbl-b. Blots were probed with anti-Flag Abs. c, Immunoprecipitation showing time-dependent recruitment of Cbl-b to TAM tyrosine kinase receptors in HeLa cells upon stimulation with His-tagged Gas6 (upper 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 450ng/ml Gas6 for 15 minutes or left untreated. Lysates were immunoprecipiated with anti-Axl antibodies. Blots were then probed using anti-Ubiquitin and anti-Axl Abs. The location of the mature form of Axl (~140kDa) is shown with an arrow. Axl, Cbl-b, and tubulin protein levels are shown to control for the input (lower 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 C373A^KI/KI NK cells. Background staining with control isotype Abs is shown for each blot in grey. f, In vitro proliferation of Cbl-b^+/- and Cbl-b^-/- NK cells stimulated with anti-NKG2D Abs (30μg/ml) 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 hours after siRNA transfection. h, Axl surface expression in siControl or siCbl-b HeLa cells stimulated with 200ng/ml recombinant human Gas6 for the indicated time periods. Western blots show efficient downregulation of Cbl-b protein levels in HeLa cells 48hrs 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, indicates the 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 1hour. 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/AlexaFluor680-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 hPBMCs) in a proliferation assay. After incubation for 72 hours 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 Ab 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 i.v. with 2.5x10⁵ B16F10 melanoma cells at day 0. At day +1 and +4, recipient mice received 1.0x10⁵ sorted syngeneic Cbl-b sufficient or Cbl-b^-/- NK cells that were either pre-treated for 2.5 hours ex vivo with vehicle (DMSO) or LDC1267 (2.5μM). A control untreated group received tumor cells but did not receive NK cells (untreated B6). Mice were euthanized at day +14 post–tumor inoculation and lung tumor foci and total tumor areas in lungs were quantified.

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

a, Kinetics of primary 4T1 tumor 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). n.s., 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 tumors and treated daily by intraperitoneally injections with vehicle or LDC1267 (20mg/kg). Data is from 12 hours after the last treatment (day +16). Horizontal line indicates median value. n.d.; not detectable. d, Plasma concentrations of LDC1267 in mice bearing 4T1 metastatic tumors treated daily by oral gavage with vehicle or LDC1267 (100mg/kg). Data is from 11 hours after the last treatment (day +21). Horizontal line indicates median value. n.d.; not detectable. e-f, Relative sizes (e) and numbers (f) of 4T1 liver micro-metastases in syngeneic mice treated with vehicle or LDC1267 (100mg/kg) via oral gavage. Mean values ± s.e.m. are shown on day +21 after initiation of LDC1267 therapy (day +27 after orthotopic tumor 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 e-f. Arrows indicate micro-metastases h, Prothrombine times (mean ± s.d.) in warfarin (0.5mg/l) and vehicle treated mice, showing that the low dose of warfarin used does not cause a coagulopathy, confirming previous results,. Of note, we never observed neither spontaneous nor excessive bleeding in warfarin-treated mice as compared to vehicle controls. Data is 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 C373A^KI/KI (n=12) and warfarin-treated Cbl-b^+/+ (n=19), Cbl-b^+/- (n=11), Cbl-b^-/- (n=9), and C373A^KI/KI (n=11) mice at day +16 post B16F10 i.v. inoculation. *P<0.05 comparing the control and warfarin-treated groups in either Cbl-b^+/+ or Cbl-b^+/- mice (Mann-Whitney test). n.s.= 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 i.v. 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 cells recovered from the peritoneal cavity 24 hrs after intra-peritoneal 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).

Figure 1. Cbl-b mutant NK cells control metastatic melanomas.

a, TC-1 tumor growth in Cbl-b^+/+Rag2^-/- and Cbl-b^-/-Rag2^-/- mice (mean ± s.e.m., n=10 each). ***P<0.001 (two-way ANOVA, Bonferroni’s post hoc test). b, Cbl-b and β-actin protein expression in Cbl-b^+/+, Cbl-b^+/-, and Cbl-b^-/- NK cells. c, IFN-γ⁺ Cbl-b^+/+ and Cbl-b^-/- NK cells (%) following anti-NKG2D stimulation. (mean ± s.e.m., n=3).***P<0.001 (two-way ANOVA, Bonferroni’s post hoc test). d, In vivo NK cell cytotoxicity towards RMA-S cells (mean ± s.e.m., n=16/10/15/14). *P<0.05, **P<0.01 (Student’s t-test). e, f, Representative lung melanoma metastases in control (e) or NK.1.1-depleted Cbl-b^+/+, Cbl-b^+/-, Cbl-b^-/-, and C373A^KI/KI mice (f) at day+21. g, B16F10 tumor-to-lung ratios (mean ± s.e.m.) of control and NK1.1⁺-depleted Cbl-b^+/+(n=12/6), Cbl-b^+/- (n=6/4), Cbl-b^-/- (n=9/4), and C373A^KI/KI (n=7/4) mice. ***P<0.001 (Student’s t-test). h, Representative B16F10 extrapulmonary metastases in a NK1.1⁺ cell depleted mouse. i, Extrapulmonary metastases in control or NK1.1-depleted mice (lines are median, day+16-21) **P<0.01***P<0.001 (Mann-Whitney test). j, k, Representative B16F10 lung metastases (j) and tumor-to-lung ratios (k) (mean ± s.e.m., day+21) in Cbl-b^+/- Prf1^+/+, Cbl-b^-/- Prf1^+/+, Cbl-b^+/- Prf1^-/- and Cbl-b^-/- Prf1^-/- mice. n=5/7/8/8. **P<0.01, ***P<0.001, n.s., not significant (One-way ANOVA, Tukey’s post hoc test).

Figure 2. Therapeutic anti-tumor activity of Cbl-b mutant NK cells.

a-d, Representative lung metastases (a), tumor-to-lung ratios (b), numbers of tumor foci (c), prevalence (d) and numbers of extrapulmonary metastases (e) at day+14 in C57BL/6J mice untreated or adoptively transferred with Cbl-b^+/+, Cbl-b^-/-, and C373A^KI/KI NK cells. For b and c, n=6/6/6/7, *P<0.05 and **P<0.001 (Mann-Whitney test). For d and e, n=16/22/14/15, *P<0.05 (Chi-square test in d and Mann-Whitney test in e). f, g, Metastases (f, H&E, asterix) and NKp46⁺ NK cells (g, arrows) in lungs of Cbl-b^+/-NeuT⁺ and Cbl-b^-/-NeuT⁺ mice. n=17/20. h,i, Representative H&E-stained lung metastases (h, arrows) and number of metastatic foci (i) in control and anti-asialo GM1-treated Cbl-b^-/-NeuT⁺ mice. n=11/10. *P<0.05 (Mann-Whitney test, lines are median). Bars, 50μM (f and g), 2000 μM (h)

Figure 3. TAM receptors are Cbl-b targets and characterization of a TAM receptor blocker.

a, In vitro Cbl-b-dependent ubiquitylation of Tyro3, Axl, and Mer (anti-Ub). Control, no TAM receptors. Loading controls are shown. b, IFN-γ⁺ Cbl-b^+/+ and Cbl-b^-/- NK cells (%) stimulated with anti-NKG2D Abs in the presence of soluble Gas6. *P<0.05 (One-way ANOVA, Dunnett’s post hoc test, n=6/5). c, TAM receptors surface expression in NK cells after stimulation with Gas6. *P<0.05 (two-way ANOVA, Bonferroni’s post hoc test, n=10-13). d, Chemical structure of the TAM receptor kinase inhibitor LDC1267. e, IC₅₀ values for the indicated protein kinases as determined by tracer assays. f, Remaining activity (compared to DMSO control) in 456 kinases treated with 1µM LDC1267 g, IFN-γ⁺ Cbl-b^+/+ and Cbl-b^-/- NK cells (%) pre-treated with vehicle or LDC1267 and then stimulated with anti-NKG2D Abs and Gas6. *P<0.05, n.s., not significant (Student’s t-test, n=11/8.). h, In vivo NK cytotoxicity towards RMA-Rae1 cells in mice treated with vehicle or LDC1267. *P<0.05, **P<0.01 (One-way ANOVA, Tukey’s post hoc test, n=16/14/9/10). i, Tumor-to-lung areas in B16F10 tumor-bearing B6 mice untreated or adoptively transplanted with NK cells ex vivo treated with vehicle or LDC1267. *P<0.05 and **P<0.01 (One-way ANOVA, Tukey’s post hoc test, n=16/16/16/10/10). Data in b,c,g-i are mean ± s.e.m.

Figure 4. Control of metastases by TAM receptor inhibition.

a-c, Lung melanoma metastases (a), tumor-to-lung ratios (b, mean ± s.e.m.), and extrapulmonary metastases (c, lines are median) in vehicle- and LDC1267-treated wild-type control or NK1.1-depleted mice. n=8 each. *P<0.05, ***P<0.001, n.s., not significant (Student’s t-test in b and Mann-Whitney test in c). d-f, Representative images (d, arrows; bars, 50μm), and numbers (e) and relative sizes (f) of 4T1 liver micro-metastasis in control or asialo-GM1 immunodepleted mice treated with vehicle or LDC1267 (20mg/kg, daily i.p.). Mean ± s.e.m., n=7/9/8/8. *P<0.05; n.s., not significant (Mann-Whitney test). g,h, Representative lung B16F10 melanoma metastases (g) and tumor-to-lung ratios (h) in vehicle and warfarin-treated (0.5 mg/l) Cbl-b^+/+ (n=12/12), Cbl-b^+/- (n=6/7), Cbl-b^-/- (n=4/7), and C373A^KI/KI (n=8/8) mice. Mean ± s.e.m.**P<0.01, *P<0.05, n.s., not significant (Student’s t-test). i, j, Relative lung tumor area (i, mean ± s.e.m.) and extrapulmonary melanoma metastases (j, lines are median) in Cbl-b^+/+ mice treated with vehicle (n=15), warfarin (n=14), vehicle and anti-NK1.1 Abs (n=5) or warfarin and anti-NK1.1 Abs (n=7). *P<0.05, ***P<0.001, n.s., not significant (One-way ANOVA, Tukey’s post hoc test for i; Mann-Whitney test for j). k, Schematic illustrating the TAM receptor/Cbl-b inhibitory pathway controlling NK cell anti-metastatic activities.