Figure 7.

Birinapant plus radiation induces tumor regression in xenografts, and model of cell death pathways modulated by birinapant. A, 2.5×10⁶ UM-SCC-46 cells were implanted into athymic nu/nu mice. Mice were randomized into four groups (vehicle control, 15 mg/kg birinapant, 10 × 2 Gy radiation, or combination, n=12 each) seven days after tumor inoculation (day 7) when average tumor volume reached ~200 mm³. Radiation beginning day 7: 2 Gy of radiation given M-F for two weeks, to 20 Gy (red double headed arrows). Birinapant beginning day 8: 15 mg/kg birinapant i.p. every three days for ten doses (purple double headed arrow). Statistical difference vs control by Student t-test (+; p < 0.05). Error bars, SEM. B, Survival analysis, significance determined by Gehan-Breslow-Wilcoxon test. Median survival, 27 days (control), 42 days (birinapant, p=0.003), 80 days (radiation, p=0.0002), and combination group without deaths, to 100 days. C, Photos of tumors of representative mice from each treatment group on day 21 (five doses of birinapant and 20 Gy radiation). D, Relative mTNFα gene expression in tumors on day 21 compared by qRT-PCR. Student t-test (*; p < 0.05). Error bars, SEM. E, The intrinsic cell death pathway, inducible by radiation DNA damage, and the extrinsic pathway, triggered by death receptor family ligands can converge on caspase-3 to induce apoptosis. Death ligand binding via the extrinsic pathway results in a cytoplasmic complex that includes FADD, which may activate caspase-8/3, or RIP1/3/MLKL-mediated signaling and necroptosis. Cell death is blocked by Inhibitors of Apoptosis Proteins (IAPs). DNA damage induced mitochondrial release of SMAC, and SMAC mimetic birinapant inhibits and degrades IAPs (cIAP1, cIAP2, XIAP) to enhance TNF-mediated cell death.