Journal: British Journal of Cancer

Article Title: YES1 amplification confers trastuzumab–emtansine (T-DM1) resistance in HER2-positive cancer

doi: 10.1038/s41416-020-0952-1

Figure Lengend Snippet: a Signalling proteins in indicated cells were determined by western blotting. BT-474, BT-474/YES1 WT and BT-474/YES1 Y537F cells ( b ), and SK-OV-3, SK-OV-3/YES1 WT and SK-OV-3/YES1 Y537F cells ( c ), were treated with different concentrations of T-DM1, trastuzumab or lapatinib for 120 h, after which cell survival was measured using sulforhodamine B assays. BT-474/YES1 Y537F ( d ) and SK-OV-3/YES1 Y537F ( e ) cells were treated with different concentrations of the HER2-targeted inhibitors, T-DM1 or lapatinib, for 120 h, with or without dasatinib (30 nM), after which cell survival was measured using sulforhodamine B assays. Data shown represent means ± SD of three independent experiments.

Article Snippet: BT-474 and SK-OV-3 cells expressing YES1 Y537F and YES1 WT were generated by transfecting cells with the YES1 Y537F (Addgene plasmid #51299) and YES1 WT (generated by point mutation from YES1 Y537F plasmid) plasmids, respectively.

Techniques: Western Blot

Journal: British Journal of Cancer

Article Title: YES1 amplification confers trastuzumab–emtansine (T-DM1) resistance in HER2-positive cancer

doi: 10.1038/s41416-020-0952-1

Figure Lengend Snippet: Mice bearing BT-474/R1-7 ( a ) or SK-OV-3/YES1 Y537F ( b ) xenograft tumours were treated with T-DM1, dasatinib, or a combination of T-DM1 and dasatinib. Tumour volumes (top) and body weights (bottom) were measured on the indicated days. Data shown represent means ± SD (error bars; control group, n = 10 or treatment groups, n = 6; * p < 0.05, ** p < 0.01, **** p < 0.0001). c BT-474/R1-7 xenograft tumours were isolated, then signalling proteins were determined by western blotting. d Proposed model of T-DM1 resistance in BT-474/R1-7 cells.

Article Snippet: BT-474 and SK-OV-3 cells expressing YES1 Y537F and YES1 WT were generated by transfecting cells with the YES1 Y537F (Addgene plasmid #51299) and YES1 WT (generated by point mutation from YES1 Y537F plasmid) plasmids, respectively.

Techniques: Control, Isolation, Western Blot

Journal: Journal of thoracic oncology : official publication of the International Association for the Study of Lung Cancer

Article Title: YES1 Is a Druggable Oncogenic Target in SCLC.

doi: 10.1016/j.jtho.2022.08.002

Figure Lengend Snippet: Figure 2. Correlation between CNV/mRNA expression and biologic effects elicited by YES1 depletion in vitro. (A) Positive correlation between YES1 mRNA expression of YES1 and CNV in a panel of 45 SCLC cell lines from the CCLE. Relative CNV corresponds to log2 CNV/2, the red line defines CNV gains/amplifications (CNV 2.5) and the blue line defines CNV deletions (CNV 0.8). (B) IHC and FISH images illustrating YES1 expression and CNV in DMS53 and H209 tumor xenografts. Scale bars: 50

Article Snippet: Silencing of YES1 was achieved with short hairpin RNAs (shRNAs) (Sigma-Aldrich), whose sequences can be found in Supplementary Table 2. shRNAs targeting YES1 (sh-YES1) were cloned into the lentiviral TetpLKO-puro plasmid (Addgene #21915). shRNA against GFP (shGFP) was used as control and lentiviral particles for both shRNAs were produced as previously described.27 Cell lines were infected by adding lentiviral particles containing the plasmid and polybrene (8 mg/ mL) and centrifuging them at 1200 revolutions per minute for 2 hours at 32oC.

Techniques: Expressing, In Vitro

Journal: Journal of thoracic oncology : official publication of the International Association for the Study of Lung Cancer

Article Title: YES1 Is a Druggable Oncogenic Target in SCLC.

doi: 10.1016/j.jtho.2022.08.002

Figure Lengend Snippet: Figure 3. Inhibition of YES1 abrogates tumor growth and metastasis. (A) Tumor growth of DMS53 shGFP (n ¼ 7) and sh-YES1 (n ¼ 7 per shRNA) cells subcutaneously implanted in immunosuppressed mice and treated with doxy when the tumors reached an initial volume of 100 mm3 to induce the shRNA expression. (B) Waterfall plot illustrating the tumor volume change of DMS53 control and YES1-inhibited tumors at day 33. (C) Mice OS curve corresponding to the experiment illustrated in A and B.

Article Snippet: Silencing of YES1 was achieved with short hairpin RNAs (shRNAs) (Sigma-Aldrich), whose sequences can be found in Supplementary Table 2. shRNAs targeting YES1 (sh-YES1) were cloned into the lentiviral TetpLKO-puro plasmid (Addgene #21915). shRNA against GFP (shGFP) was used as control and lentiviral particles for both shRNAs were produced as previously described.27 Cell lines were infected by adding lentiviral particles containing the plasmid and polybrene (8 mg/ mL) and centrifuging them at 1200 revolutions per minute for 2 hours at 32oC.

Techniques: Inhibition, shRNA, Expressing, Control

Journal: Journal of thoracic oncology : official publication of the International Association for the Study of Lung Cancer

Article Title: YES1 Is a Druggable Oncogenic Target in SCLC.

doi: 10.1016/j.jtho.2022.08.002

Figure Lengend Snippet: Figure 4. Pharmacologic inhibition of YES1 impairs cell proliferation and targets phospho-FAK tyrosine kinase. (A, B) Cell proliferation curves illustrating the IC50 for dasatinib (A) and CH6953755 (B) in DMS53, H209, and H69 cell lines. (C) H&E staining of DMS53-, H209- and PDX YU-16-derived organoids. Scale bar: 5 mm. (D–F) Growth curves of DMS53- (D), H209- (E), and YU-16-derived organoids (F) in the presence of dasatinib or CH6953755. (G) Representative images of untreated PDX YU-

Article Snippet: Silencing of YES1 was achieved with short hairpin RNAs (shRNAs) (Sigma-Aldrich), whose sequences can be found in Supplementary Table 2. shRNAs targeting YES1 (sh-YES1) were cloned into the lentiviral TetpLKO-puro plasmid (Addgene #21915). shRNA against GFP (shGFP) was used as control and lentiviral particles for both shRNAs were produced as previously described.27 Cell lines were infected by adding lentiviral particles containing the plasmid and polybrene (8 mg/ mL) and centrifuging them at 1200 revolutions per minute for 2 hours at 32oC.

Techniques: Inhibition, Staining, Derivative Assay

Journal: Journal of thoracic oncology : official publication of the International Association for the Study of Lung Cancer

Article Title: YES1 Is a Druggable Oncogenic Target in SCLC.

doi: 10.1016/j.jtho.2022.08.002

Figure Lengend Snippet: Figure 6. YES1 protein is released in exosomes and can be detected in SCLC and NSCLC plasma-derived exosomes. (A) Schematic representation of the protocol followed for the exosomes’ extraction from conditioned DMS53 and H209 culture media, plasma from the mouse models or plasma from patients with lung cancer. (B) Western blotting of the exosomal markers Alix, TSG101, CD63, and CD9, and YES1 in DMS53 and H209 cell lysates and their derived exosomes isolated from the conditioned media after 96 h. Electron microscopy images of DMS53 and H209 exosomes. (C) Western blotting illustrating the decrease in YES1 protein levels in exosomes isolated from plasmas of mice carrying shGFP (n ¼ 2), sh1-YES1 (n ¼ 1), or sh2-YES1 (n ¼ 1) H209 subcutaneous tumors. (D) Western blotting of pSFKs, and exosomal markers in plasma-derived exosomes from mice implanted with PDX YU-16 and treated with CH6953755 or dasatinib. (E) Protein expression of exosomal markers and YES1 in NSCLC patient-derived exo- somes and representative images of YES1 IHC in some of those NSCLC specimens. A representative electron microscopy image of exosomes from a patient with NSCLC is also illustrated. (F) Protein levels of the exosomal markers mentioned above and YES1 in six SCLC patient-derived exosomes and a healthy donor. Representative images of YES1 protein levels in SCLC specimens and an electron microscopy imageof exosomes isolated froma patient with SCLC. Scale barin IHC: 50mm.Scale barin exosomes images: 100 nm. h, hour; H, high; IHC, immunohistochemistry; KD, knockdown; L, low; M, medium; pSFK, phospho-SFK; sh1-YES1, shRNA 1 targeting YES1; sh2-YES1, shRNA 2 targeting YES1; shGFP, shRNAs targeting GFP; shRNA, short hairpin RNA.

Article Snippet: Silencing of YES1 was achieved with short hairpin RNAs (shRNAs) (Sigma-Aldrich), whose sequences can be found in Supplementary Table 2. shRNAs targeting YES1 (sh-YES1) were cloned into the lentiviral TetpLKO-puro plasmid (Addgene #21915). shRNA against GFP (shGFP) was used as control and lentiviral particles for both shRNAs were produced as previously described.27 Cell lines were infected by adding lentiviral particles containing the plasmid and polybrene (8 mg/ mL) and centrifuging them at 1200 revolutions per minute for 2 hours at 32oC.

Techniques: Clinical Proteomics, Derivative Assay, Extraction, Western Blot, Isolation, Electron Microscopy, Expressing, Immunohistochemistry, Knockdown, shRNA

Journal: Journal for Immunotherapy of Cancer

Article Title: SRC family kinase (SFK) inhibitor dasatinib improves the antitumor activity of anti-PD-1 in NSCLC models by inhibiting Treg cell conversion and proliferation

doi: 10.1136/jitc-2020-001496

Figure Lengend Snippet: (A) Comparison of YES1, SRC, FYN and LYN mRNA expressions between non-malignant lung tissue and NSCLC (TCGA cohort). Mann-Whitney U test was used for the statistical analysis. (B, C) Kaplan-Meier survival curves showing that high YES1 and SFK levels (above the median) are associated with worse OS in NSCLC. The log-rank test was used for the statistical comparisons. (D, E) Quantification of FOXP3+CD4+ cells in NSCLC specimens from the CUN cohort. The percentage of Tregs (FOXP3+CD4+) in YES1-positive tumors in the upper quartile of the YES1 H-score is significantly higher than that found for the rest of the tumors (D) and in the lower quartile (E). (F, G) Quantification of FOXP3+CD4+ cells in patients with LUAD from CUN. The percentage of Treg cells in the YES1 upper quartile was compared with the rest of the quartiles (F) or the lower quartile (G). (H) Representative images of YES1 IHC and multiplex immunofluorescence for CD8+, CD4+ and FOXP3+CD4+ cells in patients with NSCLC. (I) Relative abundance of Treg cells in patients with LUAD from the TCGA database analyzed with CIBERSORT tool. Scale bar: 50 µm. (D–G, I) Data are expressed as median, and statistical comparisons were performed using the Mann-Whitney U test. *P<0.05, **P<0.01. CUN, University Clinic of Navarra; IHC, immunohistochemistry; LUAD, lung adenocarcinoma; ns, not significant; NSCLC, non-small cell lung cancer; OS, overall survival; SFK, SRC family kinase; SRC, Treg, regulatory T cell.

Article Snippet: For the detection of YES1 in the TMAs by immunohistochemistry, antigen retrieval was performed by heating the samples in citrate buffer (10 mM, pH 6), and the primary anti-YES1 antibody (Proteintech, 20 243–1-AP) was incubated at 1:100 dilution.

Techniques: Comparison, MANN-WHITNEY, Multiplex Assay, Immunofluorescence, Immunohistochemistry

Journal: Journal for Immunotherapy of Cancer

Article Title: SRC family kinase (SFK) inhibitor dasatinib improves the antitumor activity of anti-PD-1 in NSCLC models by inhibiting Treg cell conversion and proliferation

doi: 10.1136/jitc-2020-001496

Figure Lengend Snippet: (A) mRNA expression of YES1, SRC, FYN and LYN analyzed by RT-qPCR in a panel of 16 murine NSCLC cell lines. (B) Western blot analysis of YES1 expression in nine murine cell lines. (C) Effect of dasatinib on 393P and UN680 cell proliferation in vitro. (D) Western blot analysis showing pSFK protein inhibition by dasatinib at 10 hours post-treatment in vitro. (E) Subcutaneous tumor growth of 393 P cells injected in athymic nude mice treated with dasatinib (30 mg/kg) or vehicle. A two-way analysis of variance followed by a post hoc Bonferroni test was used. *P<0.05, ***P<0.001. NSCLC, non-small cell lung cancer; pSFK, phospho-SRC family kinase; RT-qPCR, real-time quantitative PCR; SFK, SRC family kinase.

Article Snippet: For the detection of YES1 in the TMAs by immunohistochemistry, antigen retrieval was performed by heating the samples in citrate buffer (10 mM, pH 6), and the primary anti-YES1 antibody (Proteintech, 20 243–1-AP) was incubated at 1:100 dilution.

Techniques: Expressing, Quantitative RT-PCR, Western Blot, In Vitro, Inhibition, Injection, Real-time Polymerase Chain Reaction

Journal: Journal for Immunotherapy of Cancer

Article Title: SRC family kinase (SFK) inhibitor dasatinib improves the antitumor activity of anti-PD-1 in NSCLC models by inhibiting Treg cell conversion and proliferation

doi: 10.1136/jitc-2020-001496

Figure Lengend Snippet: (A) Schematic representation of the experimental design followed for the depletion of CD8, CD4 and NK1.1 cell populations and the treatment with dasatinib+anti-PD-1 in vivo. (B) Tumor growth of 393P-inoculated cells in the presence of depleting antibodies and treatment with dasatinib+anti-PD-1 (n=6). (C) Treg cell proliferation 4 days after plating in the presence of IL-2, anti(α)CD3, αCD28 and dasatinib (2 and 10 nM) measured by flow cytometry. (D) IL-10 levels measured in Treg cells culture medium after 48 hours of exposure to dasatinib. (E) Protein expression of pLCK, LCK, YES1, pSTAT3, STAT3, pSTAT5 and STAT5 in Treg cells treated with dasatinib (10 nM) for 45 min. (F, G) In vitro experiment of TGF-β-dependent CD4+ T cell conversion into Tregs, in the presence of IL-2, αCD3, αCD28 and dasatinib (2, 10 and 20 nM), analyzed by flow cytometry. (H) Western blotting of pSTAT5, STAT5, pSMAD3 and SMAD3 in CD4+ T cells converted into Tregs after treatment with dasatinib (10 nM). Differences between groups were evaluated with a two-way (B) or a one-way analysis of variance test (C, D, F) followed by a post hoc Bonferroni test. **P<0.01, ***P<0.001, ****P<0.0001. IL, interleukin; LCK, lymphocyte-specific protein tyrosine kinase; PD-1, programmed cell death 1; pLCK, phospholymphocyte-specific protein tyrosine kinase; TGF-β, transforming growth factor beta; Treg, regulatory T cell.

Article Snippet: For the detection of YES1 in the TMAs by immunohistochemistry, antigen retrieval was performed by heating the samples in citrate buffer (10 mM, pH 6), and the primary anti-YES1 antibody (Proteintech, 20 243–1-AP) was incubated at 1:100 dilution.

Techniques: In Vivo, Flow Cytometry, Expressing, In Vitro, Western Blot