birinapant  (MedChemExpress)

Journal: Cell Death Discovery

Article Title: RB loss sensitizes triple-negative breast cancer to apoptosis induced by cellular stress

doi: 10.1038/s41420-025-02864-4

Figure Lengend Snippet: A Correlation plot from the drug screening to depict the differential effects of birinapant, navitoclax and ABT-737 between MB157-WT and MB157-RB-del cells. B Live cell imaging approach to monitor the caspase-3/7 activity in MB157-WT and MB157-RB-del cells following treatment with different apoptotic drugs such as birinapant, navitoclax and ABT-737 at 100 nM. The error bars represent mean and SEM from triplicates. The experiment was done two independent times (***p < 0.0001 as determined by 2-way ANOVA). C Western blotting on MB157-WT and RB-del cells to examine the expression of CIAP, Bcl2 and Bax following treatment with birinapant at the indicated concentrations for 48 h. D Combination treatment involving birinapant + pemetrexed and birinapant + CHIR-124 at different concentrations in MB157-WT and RB-del cells. The synergistic interaction was determined by Synergy finder where the Bliss Synergy score was calculated. E Synergistic effect on cell death following the treatment with birinapant + pemetrexed and birinapant + CHIR-124 in MB231-WT and RB-del cells. The synergistic interaction was determined by Synergy finder where the Bliss Synergy score was calculated. F Column graphs illustrating the effect of alisertib on cell viability in MB157-WT, MB157-RB-del, MB231-WT, and MB231-RB-del cells in combination with DMSO and birinapant following five days of treatment. Error bars were determined based on mean and SD from triplicates. The experiment was done three independent times. (***p < 0.0001, *p < 0.05 as determined by student t test). G Biochemical analysis to determine the effect of alisertib + birinapant on PARP cleavage and caspase 3 cleavage in MB231-WT, MB231-RB-del, MB157-WT and MB157-RB-del cells. H Long-term colony formation assay in MB157-WT and RB-del cells following treatment with alisertib + birinapant and barasertib+birinapant for 13 days.

Article Snippet: Additional compounds, including alisertib and birinapant, were obtained from MedChem Express and similarly dissolved in DMSO to a final stock concentration of 10 mM.

Techniques: Drug discovery, Live Cell Imaging, Activity Assay, Western Blot, Expressing, Colony Assay

Journal: Cell Death Discovery

Article Title: RB loss sensitizes triple-negative breast cancer to apoptosis induced by cellular stress

doi: 10.1038/s41420-025-02864-4

Figure Lengend Snippet: A Representative immunohistochemical images on the tissues derived from MB231-WT and RB-del xenografts to validate RB expression. The scale bar represents 40 µm. B Differential effect of alisertib (10 mg/kg) in combination with birinapant (15 mg/kg) on tumor growth derived from MB231-WT and MB231-RB-del xenografts. Error bars represent mean and SEM. (***p < 0.0001 as determined by 2-way ANOVA). C Representative tumor images from MB231-WT and MB231-RB-del xenografts that were treated with vehicle or alisertib + birinapant. D . Tumor weights from MB231-WT and MB231-RB-del xenografts that were treated with vehicle or alisertib + birinapant. Error bars represent mean and SEM. (**p < 0.001 as determined by unpaired student t test). E H&E staining on the liver from tumor-bearing NSG mice treated with vehicle and alisertib+birinapant. F Effect of alisertib+birinapant on the mice body weights during the course of treatment. G Biochemical analysis to determine the in vivo effect of alisertib + birinapant in inducing cleaved PARP on the MB231-WT and MB231-RB-del xenografts. H Immunoblotting from the tumor tissues excised from mice bearing xenografts derived from MB231-WT and MB231-RB-del cells to determine the expression of CIAP, Bcl-2, Bax and cleaved caspase 3 following treatment with vehicle or alisertib + birinapant.

Article Snippet: Additional compounds, including alisertib and birinapant, were obtained from MedChem Express and similarly dissolved in DMSO to a final stock concentration of 10 mM.

Techniques: Immunohistochemical staining, Derivative Assay, Expressing, Staining, In Vivo, Western Blot

Journal: bioRxiv

Article Title: Potentiating CAR-T bystander killing by enhanced Fas/FasL signaling mitigates antigen escape in heterogeneous tumors

doi: 10.1101/2025.09.22.677496

Figure Lengend Snippet: (A) Schematic of the Fas signaling pathway highlighting key regulatory proteins and corresponding pharmacologic inhibitors. (B) Representative flow cytometry plots showing cleaved caspase-3 and Annexin V staining of CD19⁻Fas⁺ and CD19⁻Fas⁻ bystander tumor cells co-cultured with CAR-T cells in the presence or absence of birinapant (Bir). (C) Normalized viable cell counts of CD19⁻Fas⁺ and CD19⁻Fas⁻ tumor cells in mixed co-culture with CAR-T across increasing birinapant doses. n = 5 independent experiments. (D) Synergy analysis using Bliss scoring. The x-axis shows increasing effector-to-target (E:T) ratios, the y-axis shows birinapant doses, and the z-axis shows delta-scores. A Bliss synergy score >10 indicates a greater-than-additive effect. (E) Schematic of the experimental design to assess birinapant potentiation of CAR-T bystander killing in mixed-antigen tumors. (F) Survival curves for mice bearing 80:20 Fas⁺ mixed tumors treated with or without CAR-T in the presence or absence of birinapant. Mice received 2 Gy TBI on day 9 and CAR-T transfer on day 10; birinapant (15 mg/kg) was dosed twice weekly for 10 doses starting on day 9. No CAR-T cohorts, n=8 per group; CAR-T cohorts, n=28 (no birinapant) and n=27 (birinapant). Statistical significance was determined by log-rank test; 95% CI shown by shaded area above and below survival line. (G) Individual tumor growth curves for mice in (F). “No tumor” indicates the number of mice without palpable tumors at the end of the experiment, shown separately for no birinapant (top) or birinapant-treated (bottom) groups. (H) Schematic of FasL shedding by ADAM10 and its inhibition by GI254023X (GIX). (I) Surface FasL expression on CAR-T cells with or without GIX treatment in the presence of CD19 + , CD19⁻, or mixed target cell populations. (J) Normalized viable cell counts of CD19⁻Fas⁺ and CD19⁻Fas⁻ tumor cells in mixed co-culture with CAR-T across increasing GIX doses. n = 3 independent experiments. (K) Schematic of non-cleavable mutFasL resisting ADAM10-mediated shedding. (L) Schematic of retroviral vectors for αCD19 CAR constructs overexpressing either wild-type FasL (WTFasL, orange) or non-cleavable mutant FasL (mutFasL, teal). The amino acid sequence of the ADAM10 cleavage domain is shown below the constructs. (M) FasL protein expression in CAR-T cells. mutFasL migrates at slightly higher molecular weight due to non-cleavable modification. (N) Normalized viable cell counts of CD19⁻Fas⁺ and CD19⁻Fas⁻ tumor cells in mixed co-culture with WTFasL CAR-T versus mutFasL CAR-T. n = 3 independent experiments. Dots represent technical replicates, bars indicate mean ± s.e.m. Statistical significance determined by two-way ANOVA with Tukey’s multiple comparisons. *p < 0.03, **p < 0.002, ***p < 0.0002, ****p < 0.0001.

Article Snippet: The IAP inhibitor birinapant (TL32711, MedChemExpress, cat no. HY-16591) was resuspended in DMSO for a stock solution of 10mM.

Techniques: Flow Cytometry, Staining, Cell Culture, Co-Culture Assay, Inhibition, Expressing, Retroviral, Construct, Mutagenesis, Sequencing, Molecular Weight, Modification

Journal: bioRxiv

Article Title: Potentiating CAR-T bystander killing by enhanced Fas/FasL signaling mitigates antigen escape in heterogeneous tumors

doi: 10.1101/2025.09.22.677496

Figure Lengend Snippet: (A) Schematic of a CD3 + T cell engaging a CD20 + tumor cell through a CD20xCD3 bispecific antibody (BsAb) leading to Fas-mediated bystander killing on an adjacent CD20-cell. (B) Normalized viable cell counts of CD20 + , CD20⁻Fas + , and CD20⁻Fas⁻ OCI-Ly8 human lymphoma cells following co-culture with increasing doses of CD20xCD3 BsAb Epcoritamab. Bystander killing of CD20⁻Fas + cells is highlighted in red. Multiple t-tests were conducted on pre-specified comparisons, without correction. (C) Surface expression of CD19, CD20, and Fas in two human lymphoma cell lines OCI-Ly8 and Raji. (D) Mean fluorescence intensity (MFI) fold-change of surface expression in (C) . (E) Heatmap of normalized viable cell counts of OCI-Ly8 and Raji cells following 72 h co-culture with αFas agonist antibody. Boxes represent mean values. n=3 technical replicates. (F) Normalized viable cell counts of CD20 + and CD20⁻ Raji cells after 48 h co-culture with Epcoritamab in the presence or absence of birinapant. Bystander killing of CD20⁻ cells is highlighted in red. Dots indicate replicates; bars show mean ± s.e.m. Statistical significance was determined by two-way ANOVA with correction for multiple comparisons. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001.

Article Snippet: The IAP inhibitor birinapant (TL32711, MedChemExpress, cat no. HY-16591) was resuspended in DMSO for a stock solution of 10mM.

Techniques: Co-Culture Assay, Expressing, Fluorescence

Journal: bioRxiv

Article Title: Potentiating CAR-T bystander killing by enhanced Fas/FasL signaling mitigates antigen escape in heterogeneous tumors

doi: 10.1101/2025.09.22.677496

Figure Lengend Snippet: (A) Viable CD4⁺ and CD8⁺ CAR-T counts after 48 h co-culture with CD19⁺ or CD19⁻ target cells in the presence of increasing doses of birinapant. (B) Surface Fas expression on CD4⁺ and CD8⁺ CAR-T after birinapant treatment. (C) Percentage of cleaved caspase-3⁺ CD4⁺ and CD8⁺ CAR-T after co-culture ± birinapant. Increased apoptosis is observed in CD4⁺ cells. (D) Viable cell counts of CD4⁺ and CD8⁺ CAR-T after co-culture with increasing doses of birinapant. (E) Schematic of the mechanism of CD4⁺ T cell fratricide via Fas-FasL and the Fas knockout (FasKO) strategy to preserve T cell viability. (F) Surface Fas expression on scramble gRNA control (ScrKO) versus FasKO CAR-T. (G) Viable CD4⁺ and CD8⁺ CAR-T counts after co-culture with ScrKO or FasKO CAR-T in the presence or absence of birinapant. CD4 + CAR-T FasKO with birinapant is highlighted in red. n = 2 independent experiments. (H) Normalized viable cell counts of CD19⁻Fas + tumor cells following 48 h co-culture with ScrKO or FasKO CAR-T in the presence or absence of birinapant (5 µM). n = 2 independent experiments. (I) Bystander killing index of CAR-T overexpressing wild-type Fasl (WTFasL) or non-cleavable mutant FasL (mutFasL), with or without FasKO, in the presence or absence of birinapant (5 µM). Dots indicate technical replicates; bars show mean ± s.e.m. Statistical significance was determined by two-way ANOVA with Tukey’s multiple comparisons (A-D, I) or by pre-specified t-test comparisons (G-H). *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001.

Article Snippet: The IAP inhibitor birinapant (TL32711, MedChemExpress, cat no. HY-16591) was resuspended in DMSO for a stock solution of 10mM.

Techniques: Co-Culture Assay, Expressing, Knock-Out, Control, Mutagenesis

Journal: bioRxiv

Article Title: Potentiating CAR-T bystander killing by enhanced Fas/FasL signaling mitigates antigen escape in heterogeneous tumors

doi: 10.1101/2025.09.22.677496

Figure Lengend Snippet: (A) Schematic of tumor microenvironment antigen (TME-Ag)-directed bystander killing. Anti-FOLR2 CAR-T cells target FOLR2⁺ tumor-associated macrophages (TAMs), leading to Fas-dependent killing of adjacent FOLR2⁻ tumor cells. (B) Normalized viable cell counts of FOLR2⁺ Raw264.7 macrophages and FOLR2⁻ A20 tumor cells after 48 h co-culture with anti-FOLR2 CAR-T in the presence or absence of birinapant (5 µM). Bystander killing of FOLR2⁻Fas + A20 cells is highlighted in red. n = 2 independent experiments. (C) Heatmap of ID8 ovarian cancer cell viability after treatment with increasing doses of anti-Fas agonist, birinapant, or both. (D) Normalized viable cell counts of FOLR2⁺ Raw264.7 macrophages and FOLR2⁻ ID8 tumor cells after 48 h co-culture with anti-FOLR2 CAR-T in the presence or absence of birinapant (1 µM). Bystander killing of FOLR2⁻Fas + ID8 cells is highlighted in red. n = 3 technical replicates. (E) Representative flow cytometry dot plots from the co-culture experiment in (D). (F) Experimental timeline for tracking Luc + anti-FOLR2 CAR-T in mice bearing FOLR2⁻ A20 lymphoma tumors. (G) IVIS images of Luc + anti-FOLR2 CAR-T localizing to FOLR2⁻ A20 lymphoma tumor. (H) Quantification of Luc + anti-FOLR2 CAR-T from (G). Dots indicate replicates; bars show mean ± s.e.m. Statistical significance was determined by two-way ANOVA with Tukey’s multiple-comparisons test. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001.

Article Snippet: The IAP inhibitor birinapant (TL32711, MedChemExpress, cat no. HY-16591) was resuspended in DMSO for a stock solution of 10mM.

Techniques: Co-Culture Assay, Flow Cytometry

Journal: Oncology Letters

Article Title: BLID is a drug-responsive target of FOXO3a and multi-omics analysis reveals survival mechanisms and therapeutic vulnerabilities in BLID-deficient breast cancer cells

doi: 10.3892/ol.2025.15155

Figure Lengend Snippet: Validation of changes in the expression of a subset of genes in BLID-knockdown breast cancer cells. (A) PI3K-p110α protein expression was increased in BLID shRNA knockdown MCF-7 cells. (B) Left panel: cIAP2/BIRC3 protein expression was increased and DFNA5 expression was decreased in the BLID knockdown MDA-MB-231 cells in the absence or presence of Biri (50 µM; 6 h). Right panel: Reverse transcription-quantitative PCR analysis of BLID expression in MDA-MB-231 cells treated with BLID siRNA or Ctl siRNA. Gene expression was normalized to β-actin as an internal control. **P<0.01 vs. Ctl siRNA, n=3. shRNA, short hairpin RNA; siRNA, small interfering RNA; Ctl, control; Biri, birinapant; BLID, BH-3 like motif containing inducer of cell death; cIAP, cellular inhibitor of apoptosis protein; XIAP, X-linked inhibitor of apoptosis protein.

Article Snippet: Treatment with birinapant, a SMAC-mimetic, reduced cIAP1 expression in both the control and BLID knockdown cells.

Techniques: Biomarker Discovery, Expressing, Knockdown, shRNA, Reverse Transcription, Real-time Polymerase Chain Reaction, Gene Expression, Control, Small Interfering RNA