Journal: The Journal of Biological Chemistry

Article Title: BAP1 and YY1 regulate expression of death receptors in malignant pleural mesothelioma

doi: 10.1016/j.jbc.2021.101223

Figure Lengend Snippet: Expression levels of DR4 and DR5 are inversely correlated with BAP1 expression in malignant pleural mesothelioma. A , representative images of immunohistochemistry of DR4 and DR5 in a core from an MPM tissue microarray (TMA) with or without nuclear BAP1 expression (from 88 cores of 32 patients). B , semiquantitative analysis of DR4 and DR5 expression in MPM TMA cores with (n = 42) and without (n = 46) nuclear BAP1 expression. Each dot represents an average score per patient (n = 32). t Test; p = 0.024 (DR4) and p = 0.0092 (DR5). See section for details. C , immunoblots of DR4, DR5, and BAP1 protein expression in BAP1-mutant (n = 7) versus wt-BAP1 (n = 7) MPM cell lines. Duplet bands of DR5 represent two isoforms, DR5-short (DR5-S) and DR5-long (DR5-L). Sensitivity to rTRAIL treatment is indicated as font color: green sensitive (S); orange partially sensitive (PS); and red resistant (R). D , quantitative analysis of immunoblot intensity of DR4 and DR5 in wt BAP1 and BAP1-mutant MPM cell lines (DR4 t test, p = 0.046; DR5 t test, p = 0.009). Dot color indicates the sensitivity to rTRAIL treatment as shown in ( C ). E , quantitative analysis of immunoblot intensity of DR4 and DR5 in early passage MPM cells with (+) and without (−) nuclear BAP1 expression. (DR4 t test, p = 0.033; DR5 t test, p = 0.049). F , flow cytometry analysis of DR4 and DR5 cell surface expression in early passage MPM cells with (BAP1+) and without (BAP1−) nuclear BAP1 expression alongside an isotype control. BAP1, BRCA1-associated protein 1; DR, death receptor; MPM, malignant pleural mesothelioma; rTRAIL, recombinant tumor necrosis factor–related apoptosis-inducing ligand.

Article Snippet: Vectors expressing BAP1-mutant constructs were generated by site-directed mutagenesis (E0554; New England Biolabs) of the pCCL-CMV-BAP1 vector as previously described ( ).

Techniques: Expressing, Immunohistochemistry, Microarray, Western Blot, Mutagenesis, Flow Cytometry, Recombinant

Journal: The Journal of Biological Chemistry

Article Title: BAP1 and YY1 regulate expression of death receptors in malignant pleural mesothelioma

doi: 10.1016/j.jbc.2021.101223

Figure Lengend Snippet: BAP1 knockdown increases death receptor expression and TRAIL sensitivity in cancer cells. A , immunoblots of proapoptotic proteins in parental, BAP1 shRNA (shBAP1—clone 1) or empty vector (EV) shRNA transduced BAP1-wt MPM cells (H2818) across multiple time points (0, 6, 12, 24, and 48 h) post rTRAIL treatment (100 ng/ml). Duplet bands of DR5 represent two isoforms, DR5-short (DR5-S) and DR5-long (DR5-L). The bands were quantified and normalized to an average of parental cells data. B , cell viability assay of parental, shBAP1-transduced, or EV-transduced H2818 cells following treatment with a dose range of rTRAIL (0–1000 ng/ml) for 72 h. C , immunoblot analysis in BAP1-wt-clear cell renal cell carcinoma (CCRCC) cells (BB65 and Caki-1) and MPM cells (H2818) transduced with BAP1 (shBAP1 1 or shBAP1 2) or EV shRNA. The bands were quantified and normalized to EV. D , cell viability assay of EV-transduced or shBAP1-transduced wt-BAP1 CCRCC cells following treatment with a dose range of rTRAIL (0–1000 ng/ml) for 72 h. E , relative expression of DR4 and DR5 mRNA in CCRCC cells transduced with EV or shBAP1 assessed by quantitative PCR. Relative mRNA expression was normalized to beta-2-microgloblin (B2M) expression. Data shown are the mean ± SD of two experiments performed in triplicates. t test; ∗ p < 0.05, ∗∗ p < 0.01. BAP1, BRCA1-associated protein 1; MPM, malignant pleural mesothelioma; rTRAIL, recombinant tumor necrosis factor–related apoptosis-inducing ligand; TRAIL, tumor necrosis factor–related apoptosis-inducing ligand.

Article Snippet: Vectors expressing BAP1-mutant constructs were generated by site-directed mutagenesis (E0554; New England Biolabs) of the pCCL-CMV-BAP1 vector as previously described ( ).

Techniques: Expressing, Western Blot, shRNA, Plasmid Preparation, Viability Assay, Transduction, Real-time Polymerase Chain Reaction, Recombinant

Journal: The Journal of Biological Chemistry

Article Title: BAP1 and YY1 regulate expression of death receptors in malignant pleural mesothelioma

doi: 10.1016/j.jbc.2021.101223

Figure Lengend Snippet: The deubiquitinase (DUB) function of BAP1 regulates the transcription of DR4 and DR5. A , immunoblots of proapoptotic proteins in BAP1 null early passage mesothelioma cells (Meso-8T) transduced with constructs expressing wt-BAP1 (wt-BAP1), DUB-mutant BAP1 (C91A-BAP1), or a control vector (cont-vec) untreated and after 5 h of rTRAIL treatment (50 ng/ml). B , flow cytometry analysis of cell surface expression of DR4 and DR5 in Meso-8T cells transduced with constructs expressing wt-BAP1 (wt-BAP1) or one of two DUB-mutant BAP1 vectors (C91A or A95D). One-way ANOVA; ∗∗∗ p < 0.001. C , cell viability assay of Meso-8T cells transduced with wt-BAP1 or one of two DUB-mutant BAP1 vectors (C91A or A95D) following treatment with a dose range of rTRAIL (0–1000 ng/ml) for 72 h. D , relative DR4 and DR5 mRNA expression in parental Meso-8T cells and cells transduced with wt-BAP1 or C91A-BAP1. Relative mRNA expression was normalized to beta-2-microgloblin (B2M) expression. Data are shown as the mean ± SD of two experiments performed in triplicates. ∗ p < 0.05; ∗∗ p < 0.01. E , reporter assay for promoter activities of DR4 and DR5 in parental Meso-8T cells transduced with a luciferase reporter under the control of DR4 or DR5 promoter and cells further transduced with wt-BAP1 or A95D-BAP1. Firefly luciferase/Renilla luciferase ratios were determined as relative luciferase activities. Data are shown as the mean ± SD of two experiments (n = 6 in each experiment). ∗ p < 0.05; ∗∗ p < 0.01. F , cell viability assay of wt-BAP1 H2869 cells transduced with empty vector (EV) or shBAP1 following treatment with a dose range of rTRAIL (0–1000 ng/ml) for 72 h and for shBAP1 cells further transduced with DR4 (shDR4) or DR5 shRNA (shDR5) following the same treatment. G , cell viability assay of parental BAP1 null Meso-8T early passage MPM cells transduced with wt BAP1 (wt-BAP1) or DR4 (shDR4) or DR5 shRNA (shDR5) following treatment with a dose range of rTRAIL (0–1000 ng/ml) for 72 h. +, ++; lentiviral titer. Error bars represent the SD. BAP1, BRCA1-associated protein 1; rTRAIL, recombinant tumor necrosis factor–related apoptosis-inducing ligand.

Article Snippet: Vectors expressing BAP1-mutant constructs were generated by site-directed mutagenesis (E0554; New England Biolabs) of the pCCL-CMV-BAP1 vector as previously described ( ).

Techniques: Western Blot, Transduction, Construct, Expressing, Mutagenesis, Plasmid Preparation, Flow Cytometry, Viability Assay, Reporter Assay, Luciferase, shRNA, Recombinant

Journal: The Journal of Biological Chemistry

Article Title: BAP1 and YY1 regulate expression of death receptors in malignant pleural mesothelioma

doi: 10.1016/j.jbc.2021.101223

Figure Lengend Snippet: YY1 knockdown increases the expression of DRs and rTRAIL-induced cell death. A , immunoblot analysis in BAP1-wt MPM cells (H2818, MPP89, and H2591) or CCRCC cells (BB65 and Caki-1) transduced with YY1 shRNA-clone 1 (+) or an empty vector (EV) shRNA (−). Quantitative analysis of DR4 and DR5 bands from three independent experiments was performed. Average data after normalization to tubulin were shown as bar graphs. B , relative DR4 and DR5 mRNA expression in MPM cells (H2818) and CCRCC cells (Caki-1 and BB65) transduced with YY1-shRNA or EV-shRNA. Relative mRNA expression was normalized to beta-2-microgloblin (B2M) expression. Data are shown as the mean ± SD of two experiments performed in triplicates. ∗ p < 0.05; ∗∗ p < 0.01. C , cell viability assays of BAP1-wt MPM and CCRCC cells transduced with EV shRNA or shYY1 (clone 1) following treatment with a dose range of rTRAIL (0–1000 ng/ml) or MEDI3039 (0.1–100 pM) for 72 h. Error bars represent the SD. D , immunoblot analysis in BAP1-null early passage MPM Meso-8T cells and BAP1-mutant MPM cell lines (H28 and H226) transduced with YY1 (clone 1) or EV shRNA. E , cell viability assay of EV-transduced or shYY1-transduced (clone 1) cells described in D , following treatment with a dose range of rTRAIL (0–1000 ng/ml) for 72 h. BAP1, BRCA1-associated protein 1; CCRCC, clear cell renal cell carcinoma; DR, death receptor; MPM, malignant pleural mesothelioma; rTRAIL, recombinant tumor necrosis factor–related apoptosis-inducing ligand; YY1, Ying Yang 1.

Article Snippet: Vectors expressing BAP1-mutant constructs were generated by site-directed mutagenesis (E0554; New England Biolabs) of the pCCL-CMV-BAP1 vector as previously described ( ).

Techniques: Expressing, Western Blot, Transduction, shRNA, Plasmid Preparation, Mutagenesis, Viability Assay, Recombinant

Journal: The Journal of Biological Chemistry

Article Title: BAP1 and YY1 regulate expression of death receptors in malignant pleural mesothelioma

doi: 10.1016/j.jbc.2021.101223

Figure Lengend Snippet: YY1 recruits BAP1 to the promoter regions of DR4 and DR5 and represses their transcriptional activities. A , co-immunoprecipitation (co-IP) of endogenous YY1 and BAP1 in MPM (H2818 and MPP89) and CCRCC (Caki-1) cells. B , co-IP of YY1 and BAP1 in BAP1 null early passage MPM Meso-8T cells transduced with wt BAP1 (wt-BAP1) or a control vector. C , enrichment of BAP1 and YY1 in the promoter regions of DR4 and DR5. Meso-8T cells were overexpressed with wt-BAP1, catalytically inactive BAP1-mutant (C91A-BAP1) or a control vector (cont). Chromatin immunoprecipitation (ChIP) was performed against BAP1, YY1, or IgG control followed by quantitative PCR using primers specific for promoter regions of DR4 or DR5. Error bars represent the SD. p Values are calculated to compare against IgG control using Student's t test (n = 3); ∗ p < 0.05, ∗∗ p < 0.01. D , schematic model of the transcriptional regulation of TRAIL DRs by BAP1 and YY1. BAP1, BRCA1-associated protein 1; CCRCC, clear cell renal cell carcinoma; DR, death receptor; MPM, malignant pleural mesothelioma; TRAIL, tumor necrosis factor–related apoptosis-inducing ligand; YY1, Ying Yang 1.

Article Snippet: Vectors expressing BAP1-mutant constructs were generated by site-directed mutagenesis (E0554; New England Biolabs) of the pCCL-CMV-BAP1 vector as previously described ( ).

Techniques: Immunoprecipitation, Co-Immunoprecipitation Assay, Transduction, Plasmid Preparation, Mutagenesis, Chromatin Immunoprecipitation, Real-time Polymerase Chain Reaction

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: The plasmids to stably express wild-type BAP1 (pBABE-hygro-BAP1-HA, Addgene #154020), catalytically-inactive p.C91S mutant BAP1 (pBABE-hygro-BAP1-C91S-HA, Addgene #154021) or an empty vector control (pBABE-hygro, Addgene #1765) were previously described [ ].

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: The plasmids to stably express wild-type BAP1 (pBABE-hygro-BAP1-HA, Addgene #154020), catalytically-inactive p.C91S mutant BAP1 (pBABE-hygro-BAP1-C91S-HA, Addgene #154021) or an empty vector control (pBABE-hygro, Addgene #1765) were previously described [ ].

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: The plasmids to stably express wild-type BAP1 (pBABE-hygro-BAP1-HA, Addgene #154020), catalytically-inactive p.C91S mutant BAP1 (pBABE-hygro-BAP1-C91S-HA, Addgene #154021) or an empty vector control (pBABE-hygro, Addgene #1765) were previously described [ ].

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: The plasmids to stably express wild-type BAP1 (pBABE-hygro-BAP1-HA, Addgene #154020), catalytically-inactive p.C91S mutant BAP1 (pBABE-hygro-BAP1-C91S-HA, Addgene #154021) or an empty vector control (pBABE-hygro, Addgene #1765) were previously described [ ].

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: The plasmids to stably express wild-type BAP1 (pBABE-hygro-BAP1-HA, Addgene #154020), catalytically-inactive p.C91S mutant BAP1 (pBABE-hygro-BAP1-C91S-HA, Addgene #154021) or an empty vector control (pBABE-hygro, Addgene #1765) were previously described [ ].

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: The plasmids to stably express wild-type BAP1 (pBABE-hygro-BAP1-HA, Addgene #154020), catalytically-inactive p.C91S mutant BAP1 (pBABE-hygro-BAP1-C91S-HA, Addgene #154021) or an empty vector control (pBABE-hygro, Addgene #1765) were previously described [ ].

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: The plasmids to stably express wild-type BAP1 (pBABE-hygro-BAP1-HA, Addgene #154020), catalytically-inactive p.C91S mutant BAP1 (pBABE-hygro-BAP1-C91S-HA, Addgene #154021) or an empty vector control (pBABE-hygro, Addgene #1765) were previously described [ ].

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

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