Figure 5.
IAP inhibitor–induced cell death mode. (A) Schematic of the different cell death complexes induced by IAP inhibition. Cell death execution steps blocked by Q-VD-OPh, necrostatin-1, NSA, and GSK-872 are indicated. (B) Heat map showing the scaled expression of key apoptotic and necroptotic proteins measured by western blot in cell lysates of patient-derived T-PLL (n = 8) samples with varying sensitivity to IAP inhibitors. The annotation bar shows the viability (averaged over all concentrations) of each patient sample after birinapant treatment. (C) Heat map showing the correlation between protein expression (measured by western blot) of key apoptotic and necroptotic proteins and IAP inhibitor response. The Pearson correlation coefficient (R2) was computed from the viabilities of the 7 T-PLL samples with matching drug response (an eighth patient sample had protein expression, but no drug response), averaged over all concentrations. The rows and columns were arranged based on the hierarchical clustering. Dose response curves of patient-derived T-PLL (n = 11) and T-LGL (n = 1) samples showing the mean observed combination (AB) and expected combination effect according to the independent effect model (A × B, supplemental Methods) for birinapant in combination with the RIPK1 inhibitor necrostatin-1 (12.5 μM) (D), the MLKL oligomerization inhibitor NSA (2 μM) (E), the RIPK3 inhibitor GSK-872 (4 μM) (F), and the pan-caspase inhibitor Q-VD-OPh (12.5 μM) (G). Error bars indicate the standard error of the mean. (H) Scatter plot showing the relationship between measured combination effect AB (y-axis) and expected combination effect (A × B) based on the independent effect model (x-axis) for patient-derived T-PLL (n = 11) and T-LGL (n = 1) samples for necrostatin-1, NSA, GSK-872, and Q-VD-OPh. (I) Heat map showing the averaged combination effect of Q-VD-OPh (12.5 μM) and necrostatin-1 (12.5 μM) with birinapant in patient-derived T-PLL (n = 12), MCL (n = 15), CLL (n = 10), and other T-cell lymphoma (n = 10) samples. Colors show synergy (blue) and antagonism (red), indicated by adjusted P values (2-sided paired t test) for each concentration of birinapant. (J) Corresponding heat map showing the combination effect of NSA (2 μM) with birinapant in T-PLL (n = 12), MCL (n = 13), CLL (n = 20), and other T-cell lymphoma (n = 10) samples. Dose response curves of T-PLL (nT-PLL = 9) and T-LGL (nT-LGL = 1) samples showing the mean observed (AB) and expected combination effect according to the independent effect model (A × B) for birinapant in combination with the anti–TNF-α antibody adalimumab (5 μg/mL; nT-PLL = 9, nT-LGL = 1) (K), the Bruton tyrosine kinase/IL-2–inducible T-cell kinase inhibitor ibrutinib (2 μM; nT-PLL = 11, nT-LGL = 1) (L), and the TLR8 agonist motolimod (0.4 μM; nT-PLL = 11, nT-LGL = 1) (M). (N) Scatterplot showing the pairwise correlation of the CD95 surface protein expression levels (median over all malignant cells per patient) measured by spectral flow and the CI of recombinant human CD95L (5 μg/mL) and birinapant (averaged over all concentrations) in T-PLL (n = 10) and T-LGL (n = 1) samples. A trendline is indicated; 95% confidence intervals are shown as shaded gray areas. Avg. Viab., average viability; Bax, Bcl-2-associated X protein; Bid, BH3 interacting-domain death agonist; Bik, Bcl-2-interacting killer; CI, combination index; FADD, Fas associated via death domain; FLIPS, FLICE-like inhibitory protein short isoform; FLIPL, FLICE-like inhibitory protein long isoform; Expr., expression; MLKL, mixed lineage kinase domain–like pseudokinase; PARP, poly(ADP-ribose)-polymerase; RIPK1/3, receptor interacting serine/threonine kinase 1/3; TRADD, tumor necrosis factor receptor type 1 associated via death domain.

IAP inhibitor–induced cell death mode. (A) Schematic of the different cell death complexes induced by IAP inhibition. Cell death execution steps blocked by Q-VD-OPh, necrostatin-1, NSA, and GSK-872 are indicated. (B) Heat map showing the scaled expression of key apoptotic and necroptotic proteins measured by western blot in cell lysates of patient-derived T-PLL (n = 8) samples with varying sensitivity to IAP inhibitors. The annotation bar shows the viability (averaged over all concentrations) of each patient sample after birinapant treatment. (C) Heat map showing the correlation between protein expression (measured by western blot) of key apoptotic and necroptotic proteins and IAP inhibitor response. The Pearson correlation coefficient (R2) was computed from the viabilities of the 7 T-PLL samples with matching drug response (an eighth patient sample had protein expression, but no drug response), averaged over all concentrations. The rows and columns were arranged based on the hierarchical clustering. Dose response curves of patient-derived T-PLL (n = 11) and T-LGL (n = 1) samples showing the mean observed combination (AB) and expected combination effect according to the independent effect model (A × B, supplemental Methods) for birinapant in combination with the RIPK1 inhibitor necrostatin-1 (12.5 μM) (D), the MLKL oligomerization inhibitor NSA (2 μM) (E), the RIPK3 inhibitor GSK-872 (4 μM) (F), and the pan-caspase inhibitor Q-VD-OPh (12.5 μM) (G). Error bars indicate the standard error of the mean. (H) Scatter plot showing the relationship between measured combination effect AB (y-axis) and expected combination effect (A × B) based on the independent effect model (x-axis) for patient-derived T-PLL (n = 11) and T-LGL (n = 1) samples for necrostatin-1, NSA, GSK-872, and Q-VD-OPh. (I) Heat map showing the averaged combination effect of Q-VD-OPh (12.5 μM) and necrostatin-1 (12.5 μM) with birinapant in patient-derived T-PLL (n = 12), MCL (n = 15), CLL (n = 10), and other T-cell lymphoma (n = 10) samples. Colors show synergy (blue) and antagonism (red), indicated by adjusted P values (2-sided paired t test) for each concentration of birinapant. (J) Corresponding heat map showing the combination effect of NSA (2 μM) with birinapant in T-PLL (n = 12), MCL (n = 13), CLL (n = 20), and other T-cell lymphoma (n = 10) samples. Dose response curves of T-PLL (nT-PLL = 9) and T-LGL (nT-LGL = 1) samples showing the mean observed (AB) and expected combination effect according to the independent effect model (A × B) for birinapant in combination with the anti–TNF-α antibody adalimumab (5 μg/mL; nT-PLL = 9, nT-LGL = 1) (K), the Bruton tyrosine kinase/IL-2–inducible T-cell kinase inhibitor ibrutinib (2 μM; nT-PLL = 11, nT-LGL = 1) (L), and the TLR8 agonist motolimod (0.4 μM; nT-PLL = 11, nT-LGL = 1) (M). (N) Scatterplot showing the pairwise correlation of the CD95 surface protein expression levels (median over all malignant cells per patient) measured by spectral flow and the CI of recombinant human CD95L (5 μg/mL) and birinapant (averaged over all concentrations) in T-PLL (n = 10) and T-LGL (n = 1) samples. A trendline is indicated; 95% confidence intervals are shown as shaded gray areas. Avg. Viab., average viability; Bax, Bcl-2-associated X protein; Bid, BH3 interacting-domain death agonist; Bik, Bcl-2-interacting killer; CI, combination index; FADD, Fas associated via death domain; FLIPS, FLICE-like inhibitory protein short isoform; FLIPL, FLICE-like inhibitory protein long isoform; Expr., expression; MLKL, mixed lineage kinase domain–like pseudokinase; PARP, poly(ADP-ribose)-polymerase; RIPK1/3, receptor interacting serine/threonine kinase 1/3; TRADD, tumor necrosis factor receptor type 1 associated via death domain.

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