Fig 2: 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.

Fig 3: The reverse signaling of tmTNF-a promotes AICD through upregulating FasL/Fas and TRAIL/DR4(A, B, C, D) Jurkat cells were simultaneously stimulated with PHA-P (5 µg/ml) and TNF-a pAb (1:100) for 24 h. PHA-preactivated primary T cells were restimulated with aCD3 (10 µg/ml) and TNF-a pAb for 24 h. Expression of FasL/Fas (A, B) and TRAIL/DR4/DR5 (C, D) was detected by flow cytometry. (E, F) Jurkat cells transfected with 100 nM siRNA for FasL or TRAIL for 48 h were activated with PHA-P (5 µg/ml) in the absence or presence of TNF-a pAb for 24 h. The mRNA levels of FasL and TRAIL were determined by Real-time qPCR (E) and the apoptosis was detected by Annexin V/PI (F). All the quantitative data represent means ± S.D. of at least three independent experiments. *p<0.05, **p<0.01, ***p<0.001. ns: no significance.

Fig 4: BAP1-induced TRAIL resistance extends to other cancer subtypes and is dependent upon functional deubiquitinase and ASXL-binding sites.(A) BAP1-wild-type H2818, MPP-89, H2373 and H2869 MM lines were transduced with BAP1 (shBAP1) or empty vector (EV) shRNA. Immunoblot confirmed BAP1 knockdown in the BAP1 shRNA-transduced cells. Parental and transduced cells were treated with rTRAIL (1000 ng/ml) and cell viability assessed after 72 hr by MTT assay (t-test; ****p<0.0001). (B) The BAP1-wild-type breast cancer line MDAMB-231 and the renal cell carcinoma (RCC) lines Caki-1 and BB65 were transduced with BAP1 (shBAP1) or empty vector (EV) shRNA. Immunoblot confirmed BAP1 knockdown in the BAP1 shRNA transduced cells. Parental and transduced cells were treated with rTRAIL (1000 ng/ml) and cell viability assessed after 72 hr by MTT assay (t-test; ****p<0.0001). (C) The rTRAIL-sensitive H226 MM line, which harbours a homozygous deletion of BAP1, was transduced with either a GFP control, wild-type BAP1 or a mutant BAP1 containing an inactive functional domain: C91A — inactivating mutation of deubiquitinase catalytic site; ?HBM — deletion of HCF-1-binding motif; T493A — inactivating mutation of FOXK2-binding site; ?ASXL — deletion of ASXL1/2 protein-binding site; ?CTD — deletion of C-terminal domain containing nuclear localisation signal. These transduced lines were treated with 50 ng/ml rTRAIL and cell death assessed with XTT assay (one-way ANOVA; **p<0.01). (D) The parental and transduced H226 MM lines were treated with a concentration range (1–100 pM) of the small molecule death receptor agonist MEDI3039 and cell viability assessed with XTT assay. (E) The BAP1-wild-type MPP-89 MM line was transduced with ASXL1 (shASXL1), ASXL2 (shASXL2) or empty vector (EV) shRNA. qPCR confirmed a decrease in ASXL1 and ASXL2 mRNA expression in the ASXL1 shRNA and ASXL2 shRNA-transduced cells, respectively (Figure 2—figure supplement 6). Parental and transduced cells were treated with a concentration range (1–100 pM) of MEDI3039 and cell viability assessed with XTT assay. (F) Differential gene expression of apoptosis regulator genes in the catalytically inactive BAP1-mutant (C91A) relative to the wild-type BAP1-transduced (wt BAP1) H226 cells. (G) Immunoblot of apoptosis regulator proteins in the catalytically inactive BAP1-mutant (C91A), inactive ASXL1/2-binding site BAP1-mutant (?ASXL) or wild-type BAP1-transduced (wt BAP1) H226 cells. (H) Flow cytometry analysis of death receptor 4 (DR4) and 5 (DR5) cell surface expression in H226 cells transduced with the catalytically inactive BAP1-mutant (C91A) or wild-type BAP1 (wt BAP1) and of BAP1-wild-type H2818 MM cells transduced with BAP1 (KD) or empty vector (EV) shRNA. The values represent the median fluorescence intensity (MFI).

Fig 5: Synergistic effect of taxane + TRAIL treatment in LNCaP cells with DR4 and DR5 knockdown in 2D cell culture.A. Representative propidium iodide histograms of LNCaP cells treated with either 0.25 µM CBZ, 0.25 µM DTX, or 100 ng/mL TRAIL. B. Representative histograms of death receptor expression of DR4 and DR5 siRNA knockdown compared to scrambled and untreated controls. C. Cell viability of LNCaP cells after DR4 and DR5 knockdown when treated with 0.25 µM CBZ, 0.25 µM DTX, 100 ng/mL TRAIL, CBZ + TRAIL, or DTX + TRAIL and assessed via AlamarBlue assay. D. Cell viability of LNCaP cells after DR4 and DR5 knockdown when treated with 0.25 µM CBZ, 0.25 µM DTX, 100 ng/mL TRAIL, CBZ + TRAIL, or DTX + TRAIL and assessed via Annexin V/PI apoptosis assay. The values represent the mean ± SD (n = 3).