wild type bap1  (Addgene inc)

Journal: Autophagy

Article Title: Autophagy suppression via SRC induction represents a therapeutic vulnerability for BAP1 -mutant cancers

doi: 10.1080/15548627.2025.2535265

Figure Lengend Snippet: BAP1 regulates SRC transcriptionally. (A) reverse-phase protein array (RPPA) data analysis of SRC in KIRC-TCGA (clear cell renal cell carcinoma, ccRCC) stratified by BAP1 and/or PBRM1 mutation status. (B) RPPA data analysis in UVM-TCGA (uveal melanoma, UM) stratified by BAP1 mutation status. (C,D) Representative western blotting analysis of BAP1-deficient ccRCC UMRC-6 cell lines (C) and UM UPMM2 cell lines (D) reconstituted with an empty vector (EV), wild-type BAP1 , or p.C91S catalytically inactive BAP1 mutant. (E) Kaplan-Meier curves of overall survival in KIRC-TCGA depending on SRC expression levels, stratified by quartile. Intermediate SRC expression is the combination of the second and third quartiles. (F,G) qRT-PCR analysis of SRC mRNA expression in these reconstituted BAP1-deficient ccRCC (F) and UM (G) cell lines. (H) chromatin immunoprecipitation (ChIP) to assess BAP1 occupancy on the SRC promoter. Error bars represent the average ± SE ( n = 3–4). HR, hazard ratio; CI, confidence interval; n.s ., non-significant; *, p < 0.05; **, p < 0.01; ***, p < 0.001 for the indicated comparisons by t -tests.

Article Snippet: Additional validation was established by transducing the cell lines with wild-type BAP1 (pLVX-Flag-BAP1, Addgene #125840), several mutant BAP1 constructs (p.C91A, p.S10T, p.E31A, p.L49V, p.H169Q and p.G185R, Addgene #125841–4, 125847, 125849) or an empty vector control (pLVX-M-puro, Addgene #125839).

Techniques: Protein Array, Mutagenesis, Western Blot, Plasmid Preparation, Expressing, Quantitative RT-PCR, Chromatin Immunoprecipitation

Journal: Autophagy

Article Title: Autophagy suppression via SRC induction represents a therapeutic vulnerability for BAP1 -mutant cancers

doi: 10.1080/15548627.2025.2535265

Figure Lengend Snippet: BAP1 loss suppresses autophagy. The autophagic activity of UMRC-6 cells reconstituted with an empty vector (EV) or BAP1 (wild-type or p.C91S mutant) was analyzed using the following methods: (A) western blotting, (B,C) quantification of GFP-LC3 puncta ( n = 50), (D) western blotting of samples treated with Bafilomycin A1 (BafA1; 100 nM, 3 h), (E,F) quantification of autolysosomes vs . autophagosomes, (G) the number of WIPI2 dots per cell, (H) quantification of HiBiT-LC3 luminescence, and (I) the kinase activity of BECN1-bound VPS34. (J-L) Autophagy in UMRC-6 cells reconstituted with an empty vector (EV), wild-type BAP1 , or multiple BAP1 mutants observed in tumors were analyzed by microscopy of GFP-LC3 puncta formation (J,K) and western blotting (L) in cells treated with Bafilomycin A1 (100 nM, 3 h) or the DMSO control (vehicle). Error bars represent the average ± SE of three independent experiments. n.s ., non-significant; **, p < 0.01; ***, p < 0.001 for the indicated comparisons by t -tests. Non-overlapping letters (e.g., “a” vs . “b”) represent significant differences ( p < 0.05) using ANOVA and Student-Newman-Keuls test. Scale bar: 20 μm.

Article Snippet: Additional validation was established by transducing the cell lines with wild-type BAP1 (pLVX-Flag-BAP1, Addgene #125840), several mutant BAP1 constructs (p.C91A, p.S10T, p.E31A, p.L49V, p.H169Q and p.G185R, Addgene #125841–4, 125847, 125849) or an empty vector control (pLVX-M-puro, Addgene #125839).

Techniques: Activity Assay, Plasmid Preparation, Mutagenesis, Western Blot, Microscopy, Control

Journal: Autophagy

Article Title: Autophagy suppression via SRC induction represents a therapeutic vulnerability for BAP1 -mutant cancers

doi: 10.1080/15548627.2025.2535265

Figure Lengend Snippet: BAP1 loss leads to SRC-mediated suppression of autophagy. The effects of SRC inhibition on autophagy were assessed by treatment with dasatinib, and samples were analyzed by (A) western blotting, (B) HiBiT-LC3 luminescence, (C) GFP-LC3 puncta and (D) their quantification ( n = 50), (E) number of autophagosomes and autolysosomes ( n = 50), (F,G) number of GFP-FYVE puncta ( n = 50) treated with 1 µM dasatinib for 24 h or DMSO control, as well as by SRC silencing (H,I) or overexpression of a V5-tagged SRC construct (J-L). Bafilomycin A1 was added at 100 nM for 3 h and dasatinib at 1 µM for 24 h. Error bars represent the average ± SE of three independent experiments. n.s ., non-significant; *, p < 0.05; **, p < 0.01; ***, p < 0.001 for the indicated comparisons by t -tests. Non-overlapping letters represent significant differences ( p < 0.05) by ANOVA and Student-Newman-Keuls test. Scale bar: 20 μm.

Article Snippet: Additional validation was established by transducing the cell lines with wild-type BAP1 (pLVX-Flag-BAP1, Addgene #125840), several mutant BAP1 constructs (p.C91A, p.S10T, p.E31A, p.L49V, p.H169Q and p.G185R, Addgene #125841–4, 125847, 125849) or an empty vector control (pLVX-M-puro, Addgene #125839).

Techniques: Inhibition, Western Blot, Control, Over Expression, Construct

Journal: Autophagy

Article Title: Autophagy suppression via SRC induction represents a therapeutic vulnerability for BAP1 -mutant cancers

doi: 10.1080/15548627.2025.2535265

Figure Lengend Snippet: SRC binds to and phosphorylates BECN1 to regulate autophagy and proliferation. (A) BECN1 immunoprecipitation (IP) in UMRC-6 cells that overexpress SRC -V5 or an empty vector control (EV), containing plasmids for either constitutive SRC knockdown (sh SRC ) or a scrambled control (shSc). The arrow indicates the SRC -V5 band, and the bands below correspond to the heavy chain of IgG. (B) SRC -V5 was immunoprecipitated from UMRC-6 cells that overexpress SRC -V5 and were transfected with FLAG-BECN1 (or the corresponding empty vector control). (C) BECN1 (or IgG control) IP of UMRC-6 cells, followed by western blot analysis of phosphorylated tyrosine residues. (D) BECN1 IP of UMRC-6 cells overexpressing SRC -V5 (or empty vector control, EV) along with BAP1 (or EV) treated with 1 µM dasatinib for 24 h or DMSO control (vehicle). Red arrows indicate the bands for phosphorylated BECN1 (top) and VPS34 (bottom). (E,F) in vitro kinase assays of FLAG-BECN1 (or vector control) using in vitro transcribed/translated HA-SRC (E) or a kinase-dead version (F). The arrows in (E) indicate IgG heavy chains and the red asterisks (*) indicate the phosphorylated (top) and total (bottom) BECN1. (G-K) UMRC-6 cells stably expressing wild-type (WT) FLAG-BECN1, tyrosine phosphorylation mutants (non-phosphorylatable [BECN1 Y229/233/352F, BECN1-3F] or phospho-mimetic [BECN1 Y229/233/352E, BECN1-3E]) or an empty vector control (EV) were analyzed for autophagic flux by the number of GFP-LC3 puncta (G,H), the number of autolysosomes and autophagosomes (I), western blotting (J) or cell proliferation by Hoechst staining of the nuclei (K). Baf A1, 100 nM Bafilomycin A1 for 3 h; IP, immunoprecipitation; WCL, whole cell lysate. Error bars represent the average ± SE ( n = 50). n.s ., non-significant; *, p < 0.05; ***, p < 0.001 by t -tests. Non-overlapping letters represent significant differences ( p < 0.05) by ANOVA and Student-Newman-Keuls test. Scale bar: 20 μm.

Article Snippet: Additional validation was established by transducing the cell lines with wild-type BAP1 (pLVX-Flag-BAP1, Addgene #125840), several mutant BAP1 constructs (p.C91A, p.S10T, p.E31A, p.L49V, p.H169Q and p.G185R, Addgene #125841–4, 125847, 125849) or an empty vector control (pLVX-M-puro, Addgene #125839).

Techniques: Immunoprecipitation, Plasmid Preparation, Control, Knockdown, Transfection, Western Blot, In Vitro, Stable Transfection, Expressing, Phospho-proteomics, Staining

Journal: Autophagy

Article Title: Autophagy suppression via SRC induction represents a therapeutic vulnerability for BAP1 -mutant cancers

doi: 10.1080/15548627.2025.2535265

Figure Lengend Snippet: Dasatinib and SW076956 synergistically induce autophagy and reduce tumor growth in ovo and ex vivo in patient-derived tumor organoids. (A-D) Representative tumors of chorioallantoic membrane (CAM) assay (A) and quantification of tumor growth (B) of tumors treated with 10 µM Tat-BECN1 peptide (TB1) or 10 µM Tat-scrambled control (TS) (A,B) or with dasatinib (dasa, 1 µM), SW076956 (SW07, 40 µM) or a combination of both (C,D) for 7 days. (E-G) western blot analysis of several tumors treated with the TB1 or TS peptides (E), dasatinib, SW07 or a combination (F), and a representative patient-derived tumor organoid treated with the indicated compounds (G); BafA1: bafilomycin A1, 100 nM, 3 h; Das+SW07, combination of 1 µM dasatinib and 40 µM SW076956 for 24 h. Error bars represent the average ± SE. *, p < 0.05; ***, p < 0.001, t -test. (H-M) ombination effects of treatment with different concentrations of dasatinib and SW076956 on the viability of patient-derived tumor organoids (PDTOs) of ccRCC with BAP1 loss (H), ccRCC PDTOs with wild-type BAP1 (K), UM PDTOs with BAP1 loss (I,J) and UM PDTOs with wild-type BAP1 (L,M). The synergy/antagonism effects were determined using a Loewe synergy model with Combenefit software from two (I-M) or three (H) independent experiments.

Article Snippet: Additional validation was established by transducing the cell lines with wild-type BAP1 (pLVX-Flag-BAP1, Addgene #125840), several mutant BAP1 constructs (p.C91A, p.S10T, p.E31A, p.L49V, p.H169Q and p.G185R, Addgene #125841–4, 125847, 125849) or an empty vector control (pLVX-M-puro, Addgene #125839).

Techniques: In Ovo, Ex Vivo, Derivative Assay, Membrane, Chick Chorioallantoic Membrane Assay, Control, Western Blot, Software

Journal: Autophagy

Article Title: Autophagy suppression via SRC induction represents a therapeutic vulnerability for BAP1 -mutant cancers

doi: 10.1080/15548627.2025.2535265

Figure Lengend Snippet: Dasatinib and SW076956 synergistically induce autophagy and decrease cell viability in vitro . (A-D) the effects of dasatinib (at nM concentration) and SW076956 (at µM concentration) treatments on autophagy in UMRC-6 cells were assessed by western blotting alone (left panel) or in combination (right panel) (A), HiBiT-LC3 luminescence (B), and the number of GFP-LC3 puncta ( n = 50) (C,D). Cells were treated with 1 µM dasatinib and/or 40 µM SW076956 for 24 h. Baf A1, 100 nM Bafilomycin A1 for 3 h. Error bars represent the average ± SE of three independent experiments. n.s ., non-significant; *, p < 0.05; ***, p < 0.001 by t -tests. Non-overlapping letters indicate significant differences ( p < 0.05) by ANOVA and Student-newman-keuls test. (E,F) the combination effects of dasatinib and SW076956 treatments on cell viability of UMRC-6 (E) or TFK-1 (F) cells reconstituted with empty vector (EV), wild-type BAP1 , or a p.C91S BAP1 mutant were quantified 72 h after treatment of serial dilutions of single and combined compounds for three independent experiments. The synergy/antagonism effects of dasatinib and SW076956 were determined using a Loewe synergy model with Combenefit software from three independent experiments.

Article Snippet: Additional validation was established by transducing the cell lines with wild-type BAP1 (pLVX-Flag-BAP1, Addgene #125840), several mutant BAP1 constructs (p.C91A, p.S10T, p.E31A, p.L49V, p.H169Q and p.G185R, Addgene #125841–4, 125847, 125849) or an empty vector control (pLVX-M-puro, Addgene #125839).

Techniques: In Vitro, Concentration Assay, Western Blot, Plasmid Preparation, Mutagenesis, Software

Journal: Autophagy

Article Title: Autophagy suppression via SRC induction represents a therapeutic vulnerability for BAP1 -mutant cancers

doi: 10.1080/15548627.2025.2535265

Figure Lengend Snippet: Schematic representation of the proposed mechanism and platform for stratifying BAP1-loss patients who could benefit from treatment with SRC inhibitors and autophagy inducers. Lethal cancers with BAP1 mutations suppress autophagy through the binding and phosphorylation of BECN1 by the proto-oncogene SRC. Treatments with SRC inhibitors and autophagy inducers exhibited synergism in vitro , in ovo and ex vivo in patient-derived tumor organoids (PDTOs) with BAP1 loss, paving the way for the treatment of BAP1-deficient cancers with a combination of autophagy inducers and kinase inhibitors. The BAP1 immunohistochemistry images were reproduced from [10] with permission from Springer Nature.

Article Snippet: Additional validation was established by transducing the cell lines with wild-type BAP1 (pLVX-Flag-BAP1, Addgene #125840), several mutant BAP1 constructs (p.C91A, p.S10T, p.E31A, p.L49V, p.H169Q and p.G185R, Addgene #125841–4, 125847, 125849) or an empty vector control (pLVX-M-puro, Addgene #125839).

Techniques: Binding Assay, Phospho-proteomics, In Vitro, In Ovo, Ex Vivo, Derivative Assay, Immunohistochemistry

Journal: Molecular Cancer Research

Article Title: Multi-omics Profiling Shows BAP1 Loss Is Associated with Upregulated Cell Adhesion Molecules in Uveal Melanoma

doi: 10.1158/1541-7786.mcr-21-0657

Figure Lengend Snippet: Figure 1. BAP1 mutations are correlated with increased expression of CAMs CDH1, CADM1, and SDC2. A, On the basis of BAP1 mutation and copy loss, TCGA uveal melanoma samples were strati- fied into BAP1 mutant and wild-type groups. Differential expression analy- sis was performed between BAP1 mutant (n ¼ 40) and wild-type (n ¼ 40) samples and used for performing GSEA. GSEA enrichment plots of the Hallmark epithelial to mesenchymal and KEGG CAMs) gene sets in BAP1 mutant versus wild-type groups are shown. B, A heatmap showing z-score values for the top 10 genes from the Hallmark EMT and KEGG CAMs gene sets in TCGA uveal mela- noma patient tumor samples. C, CDH1, CADM1, and SDC2 gene expression from TCGA RNA-seq data in BAP1 wild-type (n ¼ 40) and mutant sam- ples (n ¼ 40). D, Analysis of TCGA data for uveal melanoma patient progres- sion-free survival according to CDH1, CADM1, and SDC2 expression, strati- fied by high or low median RNA expression. Log-rank test was used to determine the significance of progres- sion-free survival (https://wiki.nci.nih. gov/plugins/servlet/mobile#content/ view/24279961).

Article Snippet: For BAP1 expression, wild-type BAP1 plasmid, pLVX-M-Flag-BAP1 was used; it was a gift from Boyi Gan (Addgene plasmid # 125840; http://n2t.net/addgene:125840; RRID: Addgene_125840; ref. 6).

Techniques: Expressing, Mutagenesis, Quantitative Proteomics, Gene Expression, RNA Sequencing, RNA Expression

Journal: Molecular Cancer Research

Article Title: Multi-omics Profiling Shows BAP1 Loss Is Associated with Upregulated Cell Adhesion Molecules in Uveal Melanoma

doi: 10.1158/1541-7786.mcr-21-0657

Figure Lengend Snippet: Figure 2. BAP1 mutant and wild-type uveal melanoma cell line panel shows dif- ferential expression of CDH1, CADM1, and SDC2. Differential expression analysis of RNA-seq data from tripli- cates of six BAP1 mutant (MM28, MP38, MP46, MP65, WM3618F, and PDX4) and two BAP1 wild-type cell lines (92.1 and MM66) was performed using DESeq2. A, A heatmap showing average expression of six BAP1 mu- tant and two wild-type cell lines for the top 10 positively enriched KEGG CAMs pathway genes. B, Box plots of CDH1, CADM1, and SDC2 mRNA expression in BAP1 mutant (n ¼ 18) versus wild-type (n ¼ 6) cell line samples. C, A volcano plot showing antibodies targeting genes from the KEGG CAM pathway from the RPPA data. D, A heatmap of median- centered, log2-transformed RPPA data for the top and bottom five proteins ranked by the product of the fold change and log10(Benjamini– Hochberg FDR), when comparing BAP1 mutant versus wild-type cell lines. Each cell line was done in triplicate. Proteins are ordered on the basis of hierarchical clustering. E, Uveal melanoma cell line panel showing expression of E-cadherin and CADM1 in BAP1 wild-type versus mutant cell lines. F, Dot plot showing the average expression and percent of cells expressing CDH1, CADM1, and SDC2 from patient tumor scRNA-seq data. Cells were separat- ed into nonmalignant and tumor- specific malignant cell groups.

Article Snippet: For BAP1 expression, wild-type BAP1 plasmid, pLVX-M-Flag-BAP1 was used; it was a gift from Boyi Gan (Addgene plasmid # 125840; http://n2t.net/addgene:125840; RRID: Addgene_125840; ref. 6).

Techniques: Mutagenesis, Expressing, Quantitative Proteomics, RNA Sequencing, Transformation Assay

Journal: Molecular Cancer Research

Article Title: Multi-omics Profiling Shows BAP1 Loss Is Associated with Upregulated Cell Adhesion Molecules in Uveal Melanoma

doi: 10.1158/1541-7786.mcr-21-0657

Figure Lengend Snippet: Figure 3. Changes in CAMs gene expression due to BAP1 reexpression. A, Scatter plots showing fold change mRNA expression values for BAP1 mutant versus BAP1 reexpression or BAP1 mutant versus BAP1 wild-type com- parisons from four independent datasets (14, 53) for the KEGG CAMs gene set. Red dots indicate genes with log2 fold changes greater than 1 and BHFDR value of less than 0.05. B, A BAP1-mutant uveal melanoma cell line (53) was transfected with wild-type or mutant BAP1 to pro- duce proficient and deficient reex- pressing samples, respectively. Each of the mutant reexpressing and parental samples were compared against the wild-type reexpressing samples. Rank list plots showing the fold change values for genes in the KEGG CAMs pathway for parental (left), mutant A95P reexpressing (middle), and mutant C91W reex- pressing (right) samples compared against BAP1 wild-type reexpressing samples. C, Expression of BAP1 in BAP1 mutant cell cline, MP46, was confirmed by Western blot analysis, and protein expression of E-cadherin decreased in MP46-BAP1 cells. MP46-GFP was used as a control. Representative Western blots from triplicate experiments are shown.

Article Snippet: For BAP1 expression, wild-type BAP1 plasmid, pLVX-M-Flag-BAP1 was used; it was a gift from Boyi Gan (Addgene plasmid # 125840; http://n2t.net/addgene:125840; RRID: Addgene_125840; ref. 6).

Techniques: Gene Expression, Expressing, Mutagenesis, Transfection, Western Blot, Control