Anti-CD3/birinapant-induced cell death is dependent on RIPK1. (A) Western blot analysis of IAP1 and IAP2 expression and RIPK1 phosphorylation in CEM T-ALL cells stimulated for 4 hours with anti-CD3, birinapant (2.5 nM), or the anti-CD3/birinapant combination. RIPK1 and actin expression are used as normalizers. (B) CEM T-ALL cells were either left untreated (blue), or treated for 24 hours with either anti-CD3 (red), birinapant (2.5 nM, green), or the anti-CD3 + birinapant combination (orange), in either the absence (bright columns) or presence (faded columns) of the RIPK1 inhibitor necrostatin 2. Cell death (PI⁺ cells) was measured by flow cytometry, 24 hours after start of the treatment. The average of 3 independent experiments is shown. (C) Western blot analysis of RIPK1 expression in control (−) vs CEM T-ALL cells stably expressing a RIPK1 short-hairpin RNA (+). Actin is used as a loading control. (D) Percentage of cell death as measured by PI positivity by flow cytometry in nontreated cells (blue), or 24 hours after treatment with either anti-CD3 (red) or birinapant (100 nM; green) or the combination of anti-CD3 + birinapant (orange). The average of 3 independent experiments is shown. (E) NSG mice were injected with 10⁶ UPNT420 T-ALL cells. At high leukemia burden, mice were treated with either control IgG2a/carrier solvent (blue dots), or 4 μg OKT3/carrier solvent (red squares), or the combination of OKT3/birinapant/carrier solvent (green triangles), or the same combination together with the RIPK1 inhibitor (50 mg/kg, orange triangles) for 12 hours, after which time leukemia burden (hCD45⁺/hCD7⁺ cells), caspase 3/7 and caspase 8 activities were analyzed by flow cytometry. Ctl, control. ∗P < 0.05; ∗∗P < 0.01; ∗∗∗P < 0.001.