Fig. 4
Effect of talazoparib in combination with GO against AML cell lines. A Heatmaps showing the effect on cell viability of subtoxic concentrations of GO in combination with talazoparib for 72 h in AML cells. In the heatmaps, the color scale represents the values of mean normalized cell viability (% of cell viability relative to control) of at least three independent experiments. The white number inside each square of the heatmaps represents the combination index (C.I) values. C.I < 1 means synergism; C.I = 1–1.3 means additivity; C.I > 1.3 means antagonist; NC = combination index Not Calculable using Compusyn software. B Two-dimensional synergy map of AML cell lines treated with increasing concentrations of GO and talazoparib for 72 h. Zero Interaction Potency (ZIP) score is expressed as δ value. C Reduction of cell viability of AML cell lines treated with γ-calicheamicin (OCI-AML3 = 150 pM; KG-1 = 70 pM; MV4-11 = 150 pM; MOLM-13 = 4.7 pM; KASUMI-1 = 1.9 pM) in combination with olaparib (OCI-AML3 = 50 μM; KG-1 = 100 μM; MV4-11 = 200 μM; MOLM-13 = 18 μM; KASUMI-1 = 32 μM) or veliparib (OCI-AML3 = 100 μM; KG-1 = 100 μM; MV4-11 = 100 μM; MOLM-13 = 27 μM; KASUMI-1 = 42 μM) for 24 h. Histograms represent the mean ± standard deviation of normalized cell viability of three independent experiments. D Histograms represent the percentage of Annexin V + cells of AML cell lines treated with GO (OCI-AML3 = 1473 ng/mL; KG-1 = 1473 ng/mL; MV4-11 = 1473 ng/mL; MOLM-13 = 1.97 ng/mL; KASUMI-1 = 1.97 ng/mL) and talazoparib (OCI-AML3 = 3 μM; KG-1 = 3 μM; MV4-11 = 3 μM; MOLM-13 = 3 μM; KASUMI-1 = 3 μM) for 48 h (results of at least three independent experiments). E Histograms represent the percentage of cells in different cell-cycle phases after 18, 24 and 48 h of treatment with GO (OCI-AML3 = 1473 ng/mL; KG-1 = 1473 ng/mL; MV4-11 = 1473 ng/mL; MOLM-13 = 1.97 ng/mL; KASUMI-1 = 1.97 ng/mL) and/or talazoparib (OCI-AML3 = 3 μM; KG-1 = 3 μM; MV4-11 = 3 μM; MOLM-13 = 3 μM; KASUMI-1 = 3 μM). Histograms represent mean and standard deviation of at least three independent experiments. F Histograms represent the variation of G2/M and S phase cells between GO in combination with talazoparib and cells treated with GO alone. Positive fold-change (FC) indicates increased values while negative FC indicates decreased values. Asterisks indicate statistical significance of each condition compared to control cells. G Immunoblot analysis of AML cells treated with GO (OCI-AML3 = 1473 ng/mL; KG-1 = 1473 ng/mL; MV4-11 = 1473 ng/mL; MOLM-13 = 1.97 ng/mL; KASUMI-1 = 1.97 ng/mL) and talazoparib (OCI-AML3 = 3 μM; KG-1 = 3 μM; MV4-11 = 3 μM; MOLM-13 = 3 μM; KASUMI-1 = 3 μM) for 24 h. β-actin was used for loading normalization. In the lower part of the figure histograms represent the average signal obtained from relative band quantification of at least three independent experiments. H Histograms showing the percentage of pH2AX+/pMPM2+ cells in AML cell lines treated with GO (KASUMI-1 = 1.97 ng/mL, MOLM-13 = 1.97 ng/mL, OCI-AML3 = 1473 ng/mL, MV4-11 = 1473 ng/mL and KG-1 = 1473 ng/mL) for 48 h and with talazoparib (IC50 values after 24 h of treatment) for additional 6 h. I Comet assay in OCI-AML3 cell line treated with γ-calicheamicin (IC50 value) in combination with talazoparib (IC50 value) for 18 h. In the lower part of the figure scatter-plots with histograms show the variation of Tail Area, Tail Length and Tail moment between controls and treatments. J Clonogenic results of MV4-11, KG-1, OCI-AML3, MOLM-13 and KASUMI-1 cells treated with sub-toxic concentrations of γ-calicheamicin (OCI-AML3: 5 pM; KG-1: 7.5 pM; MV4-11: 2 pM; MOLM-13: 0.3 pM; KASUMI-1:0.02 pM) and talazoparib (OCI-AML3: 0.166 μM; KG-1: 0.05 μM; MV4-11: 0.33 μM; MOLM-13: 0.06 μM; KASUMI-1:0.11 μM) for 10–14 days. K Clonogenic results of MV4-11, KG-1, OCI-AML3, MOLM-13 and KASUMI-1 cells treated with sub-toxic concentrations of GO (OCI-AML3 = 1473 ng/mL; KG-1 = 1473 ng/mL; MV4-11 = 1473 ng/mL; MOLM-13 = 1.97 ng/mL; KASUMI-1 = 1.97 ng/mL) and talazoparib (OCI-AML3: 0.166 μM; KG-1: 0.05 μM; MV4-11: 0.33 μM; MOLM-13: 0.06 μM; KASUMI-1:0.11 μM) for 10–14 days